BCL-xγ, a novel BCL-x isoform, and uses related thereto

ABSTRACT

The present invention relates to BCL-xγ, a novel isoform of the BCL-x family of proteins which is predominantly expressed in T-lymphocytes and is associated with resistance to apoptosis. Both compositions of matter and methods are described which are useful in the treatment or prevention of immune system disorders.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 08/899,367filed on Jul. 23, 1997 now U.S. Pat. No. 6,472,170 which claims thebenefit of Provisional Application No. 60/023,666 filed Aug. 2, 1996.The contents of all of the aforementioned application(s) are herebyincorporated by reference.

GOVERNMENT FUNDING

Work described herein was funded, in part, by one or more grants awardedby the National Institutes of Health. The U.S. Government, therefore,may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Members of the family of BCL-2-related proteins serve as regulators ofprogrammed cell death, or apoptosis (Cory, 1995 Annu. Rev. Immunol. 13,513–543; Hockenbery, 1995 Nature 348, 334–336; Nunez et al., 1994 Nature348, 334–336; Reed, 1994. J. Cell Biol. 124, 1–6; Akbar et al., 1993.Immunology Today 14, 526–532). Apoptosis has been shown to be involvedin several immune processes, including intrathymic deletion ofautoreactive cells, elimination of peripheral T cells during theresponse to viral and bacterial superantigens and lysis of target cellsby cytotoxic T lymphocytes. There is increasing evidence that clonalexpansion of antigen-specific T-cells is determined by the relativelevel of cellular proliferation and apoptosis following TCR ligation(Zinkemagel et al. 1993. Immunol. Rev. 131:199). However, the geneticmechanisms responsible for regulating these response phenotypes are notwell understood.

The first gene to be identified which encoded a protein in this family,bcl-2, was cloned from the chromosomal breakpoint of t(14;18)-bearingB-cell lymphomas (Tsujimoto et al., 1984. Science 226:1097) and shown toinhibit cellular susceptibility to apoptosis (Cory, supra).

Several genes with homology to bcl-2 have subsequently beencharacterized, including the following: A1, which encodes an 80-aminoacid protein that is rapidly induced in macrophages in response toGM-CSF or LPS (Lin et al., 1993. J. Immunol. 151, 1979–1988); MCL1, anearly response gene in myeloid cell lines which undergo macrophagedifferentiation (Kozopas et al., 1993. Proc. Natl. Acad. Sci. USA 90,3516–3520); and Bak, a BCL-2 homologue that may enhance apoptosis(Chittenden et al., 1995. Nature 374:733; Kiefer et al., 1995. Nature374:736).

The bcl-x gene product, closely related to the BCL-2-related proteinfamily, also protects cells from apoptosis. Analysis of mice deficientin BCL-x has suggested that its function is to support the viability ofimmature cells during development of the nervous and hematopoieticsystems (Motoyama et al., 1995. Science 267, 1506–1510; Ma et al., 1995.Proc. Natl. Acad. Sci. USA 92, 4763–4767). Alternative splicing of humanbcl-x may result in at least two distinct BCL-x mRNA species. Thepredominant protein product (233 amino acids) of the larger BCL-x mRNA,BCL-xL, inhibits cell death upon growth factor withdrawal (Boise et al.,1993. Cell 74, 597–608) and its transgenic expression alters thymocytematuration leading to increased numbers of mature thymocytes (Chao etal., 1995. J. Exp. Med. 182, 821–828; Grillot et al., 1995. J. Exp. Med.182, 1973–1983). After coligation of CD3 and CD28 in murine T-cells,enhanced BCL-xL expression may confer protection from apoptosis (Boiseet al., 1995. Immunity 3, 87–98; Radvanyi et al., 1996. J. Immunol. 156,1788–1798; Mueller et al., 1996. J. Immunol. 156, 1764–1771). Thecontribution of other isoforms of this gene to activation-induced deathin T-cells is less well-defined (Gonzalez-Garcia et al., 1994.Development 120, 3033–3042; Fang et al., 1994 J. Immunol. 153,4388–4398). A second human BCL-x isoform, BCL-xS, encodes a smallerprotein of 170 amino acids which may enhance apoptosis, suggesting thatdifferent members of the BCL-x family may have opposing functions.Additional murine BCL-x isoforms, termed BCL-xβ and BCL-xΔTM, have beendefined. The β isoform may inhibit apoptosis in neurons (Gonzalez-Garciaet al., 1995. Proc. Natl. Acad. Sci. U.S.A. 92, 4304–4308) and the ΔTMisoform may inhibit apoptosis in B-cells (Fang et al., supra).

Several proteins which interact with BCL-2 proteins have also beenidentified including bax, Nip1, Nip2, Nip 3, bad, and bag-1. Thesevarious BCL-2 binding proteins have different effects on apoptosis. Forexample, bak and bax function as inducers of apoptosis, whereas bagincreases the resistance of cells to apoptosis (Farrow and Brown. 1996.Curr. Opin. Genetics and Devel. 6:45).

Despite the apparent importance of BCL-x in development and function ofT-cells, none of the BCL-x isoforms described so far displays restrictedexpression with respect to this lineage; all four isoforms of BCL-x areubiquitously expressed in a wide variety of tissues (Gonzalez-Garcia etal., 1994. Development 120, 3033–3042; Fang et al., supra). This may bebecause previous studies have isolated most of BCL-x isoforms (BCL-xL,BCL-xS and BCL-xΔTM) after screening cDNA libraries from tissues otherthan T-cells (Gonzalez-Garcia et al., 1994 supra; Fang et al., supra).The physiologic expression of these BCL-x isoforms is not sufficient toconfer resistance to apoptosis following TCR ligation, since they areexpressed equally well in apoptotic and non-apoptotic T-cell blasts.Moreover, overexpression of Bcl-xL does not affect thymocyte selection(Grillot et al. 1995. J. Exp. Med. 182:1973).

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as “BCL-xγ” nucleic acid and proteinmolecules. The BCL-xγ molecules of the present invention are useful asmodulating agents in regulating a variety of cellular processes.

Analysis of the BCL-xγ protein indicates that it contains a BH1 and aBH2 domain which are found in other BCL-x family members. However, theBCL-x γ protein also contains a novel γ domain (shown in amino acids185–235 of SEQ ID NO: 2, which includes an ankyrin domain, e.g., aminoacids 185–217 of SEQ ID NO: 2). The γ domain (C-terminal amino acids185–235) of the deduced BCL-xγ protein lacks homology with the C-terminiof previously described murine BCL-xγ isoforms, including BCL-xL, BCL-xβor BCL-xΔTM. Since BCL-xγ does not contain an apparent hydrophobicdomain flanked by charged residues it is unlikely to be membrane-bound,similar to the murine BCL-xΔTM isoform (Gonzalez-Garcia et al., 1994,supra; Fang et al., supra) but in contrast to both, human and murine,BCL-xL and BCL-xS isoforms (Boise et al., 1993) whose C-termini containsequences that may serve as membrane-anchoring domains (Chen-Levy etal., (1989) Mol. Cell Biol. 9, 701–710; Hochenbery et al., 1990 348,334–336; Nguyen et al., 1993 J. Biol. Chem. 268, 25265). The BCL-x γprotein has a calculated molecular weight of approximately 26,122 andmigrates at approximately 33 kD. The murine amino acid sequence is shownin Seq. ID No. 2.

In contrast to BCL-xL, BCL-xβ, and BCL-xΔTM, which are expressed in alltissues tested, including brain, eyes, intestine, kidney, liver, lung,lymph nodes, and thymus, the BCL-xγ isoform was detected selectively inthymus, lymph nodes, lung, and eye, but not in heart, intestine, kidney,liver, or brain. BCL-xγ has been found to be expressed only inT-lymphocytes since its message is detected in lymph nodes from BALB/ccontrol but not from BALB/c nu/nu mice or from Rag-2 deficient mice.BCL-xγ is expressed in the less mature, cortisone-sensitive fraction ofthymocytes. In addition, BCL-xγ has not been detected in thymuses fromclass I or class II MHC-deficient B6 mice, implying that expression ofthis Bcl-x isoform may normally depend on an interaction between the TCRand MHC/peptide complexes. The fact that BCL-xγ has been detected indouble positive thymocytes indicates that it plays a role in thymicselection not played by other BCL molecules. Thus, unlike previouslydescribed forms of BCL-x molecules, BCL-x γ proteins of the inventionare specifically connected to TCR ligation and are essential forresistance to TCR-dependent apoptosis.

In one aspect, the invention features an isolated nucleic acid moleculecomprising a nucleotide sequence encoding a naturally occurring BCL-xγ.In one embodiment a BCL-xγ nucleic acid molecule encodes mouse BCL-xγ.In another embodiment a BCL-xγ nucleic acid molecule encodes humanBCL-xγ. In a preferred embodiment an isolated BCL-xγ nucleic acidmolecule encodes the amino acid sequence of SEQ ID NO: 2.

In one embodiment a BCL-xγ nucleic acid molecule comprises a nucleotidesequence encoding a protein having an amino acid sequence at least 60%homologous to the γ domain shown in amino acids 185–235 of SEQ ID NO: 2and having anti-apoptotic activity.

In one embodiment a BCL-xγ nucleic acid molecule is at least 92%homologous to the nucleic acid sequence shown in SEQ ID NO:1 or acomplement thereof. In a preferred embodiment, a BCL-xγ nucleic acidmolecule comprises the sequence shown in SEQ ID NO: 1.

In another embodiment a BCL-xγ nucleic acid molecule encodes anintracellular protein which is anti-apoptotic and has an ankyrin-likedomain.

In another embodiment a BCL-xγ nucleic acid molecule comprises anucleotide sequence at least 80% homologous to the nucleotide sequenceshown in nucleotides 930–1082 of SEQ ID NO:1 or a complement thereof.

In another embodiment a BCL-xγ nucleic acid molecule specificallydetects a BCL-xγ nucleic acid molecule relative to a nucleic acidmolecule encoding another BCL-x molecule. For example, in one embodimenta BCL-xγ nucleic acid molecule hybridizes under stringent conditions toa nucleic acid molecule comprising the nucleotide sequence shown innucleotides 930–1082 of SEQ ID NO: 1.

In a preferred embodiment an isolated BCL-xγ nucleic acid moleculecomprises the coding region of SEQ ID NO: 1 or a complement thereof. Inanother embodiment a BCL-xγ nucleic acid molecule further comprisesnucleotides 1083–1384 of SEQ ID NO:1. In yet another embodiment a BCL-xγnucleic acid molecule further comprises nucleotides 1–164 of SEQ IDNO:1. In a further embodiment a BCL-xγ nucleic acid molecule furthercomprises one or more of: domain B, represented by SEQ ID NO:3; domainC, represented by nucleotides 1085–1193 of SEQ ID NO:1; domain D,represented by SEQ ID NO:4; and domain E, represented by nucleotides1194–1384 of SEQ ID NO:1 downstream of the BCL-xγ coding sequence. Inyet another embodiment a nucleic acid molecule of the present inventionhas a transcriptional regulatory sequence comprising nucleotides 1–164of SEQ ID NO:1, which may be operatively linked to the BCL-xγ codingsequence or a heterologous coding sequence.

In yet another embodiment the invention provides an isolated nucleicacid molecule which is antisense to the coding strand of a BCL-xγnucleic acid molecule

Another aspect of the invention provides a vector comprising a BCL-xγnucleic acid molecule. In certain embodiments the vector is arecombinant expression vector. In another embodiment the inventionprovides a host cell containing a vector of the invention. The inventionalso provides a method for producing BCL-xγ protein by culturing a hostcell of the invention in a suitable medium until BCL-xγ protein isproduced.

In another aspect the invention provides isolated or recombinant BCL-xγproteins. In one embodiment a BCL-xγ protein has an ankyrin-like domain,is intracellular, and is anti-apoptotic. In another embodiment anisolated BCL-xγ protein has (i) an amino acid sequence at least 60%homologous to the γ domain amino acid sequence shown in amino acids185–235 of SEQ ID NO: 2 and (ii) having anti-apoptotic activity. In apreferred embodiment a BCL-xγ protein has the amino acid sequence of SEQID NO: 2. In another embodiment of the invention a BCL-xγ protein is atleast about 83.5% homologous to the protein shown in SEQ ID NO:2.

In another embodiment the invention provides a BCL-xγ fusion protein

In another aspect of the invention, antibodies that specifically bindBCL-xγ protein are provided. In one embodiment, the antibodies of thepresent invention are monoclonal. In another embodiment the subjectantibodies are polyclonal.

In another aspect, the invention provides a nonhuman transgenic animalwhich contains cells carrying a transgene encoding BCL-xγ protein. Inone embodiment the transgene alters an endogenous gene encodingendogenous BCL-xγ protein.

In another aspect the present invention provides a method for detectingthe presence of BCL-xγ activity in a biological sample by contacting thebiological sample with an agent capable of detecting an indicator ofBCL-xγ activity such that the presence of BCL-xγ activity is detected inthe biological sample. In one embodiment the agent detects BCL-xγ mRNA,e.g., a labeled nucleic acid probe capable of hybridizing to BCL-xγmRNA. In another embodiment the agent detects BCL-xγ protein, e.g., alabeled antibody that specifically binds to BCL-xγ protein.

In another aspect, the invention provides a method for modulating BCL-xγactivity in a cell comprising contacting the cell with an agent thatmodulates BCL-xγ activity such that BCL-xγ activity in the cell ismodulated. In one embodiment, the agent inhibits BCL-xγ activity. Inanother embodiment, the agent stimulates BCL-xγ activity. In a preferredembodiment an agent modulates apoptosis in a cell. In one embodiment theagent is an antibody that specifically binds to BCL-xγ protein. Inanother embodiment the agent modulates transcription of a BCL-xγ gene ortranslation of a BCL-xγ mRNA. In yet another embodiment, the agent is anucleic acid molecule having a nucleotide sequence that is antisense tothe coding strand of the BCL-xγ mRNA or the BCL-xγ gene.

In one embodiment, the methods of the present invention are used tomodulate apoptosis in a T cell. Such methods can be used, e.g., to treatan immune system disorder. In one embodiment BCL-xγ activity isdownmodulated to ameliorate an autoimmune disorder. In anotherembodiment BCL-xγ activity is upmodulated to ameliorate animmunodeficiency.

The present invention also provides a diagnostic assay for identifying acell or cells at risk for apoptosis in a cell sample, the presence orabsence of a genetic lesion characterized by at least one of (i)aberrant modification or mutation of a gene encoding a BCL-xγ protein,and (ii) mis-regulation of said gene; (iii) aberrant post-translationalmodification of a BCL-xγ protein, wherein a wild-type form of said geneencodes an protein with a BCL-xγ anti-apoptotic activity.

In another aspect the invention provides a method for identifying acompound that modulates the anti-apoptotic activity of a BCL-x γprotein, by providing a indicator composition comprising a BCL-x γprotein having BCL-x γ anti-apoptotic activity, contacting the indicatorcomposition with a test compound, and determining the effect of the testcompound on BCL-x γ anti-apoptotic activity in the indicator compositionto identify a compound that modulates the anti-apoptotic activity of aBCL-x γ protein.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is the murine cDNA sequence (SEQ ID NO: 1) and predicted proteinsequence (SEQ ID NO: 2) of BCL-xγ (GenBank access number U51277).

FIG. 1B is a schematic comparison of murine BCL-x isoforms: BCL-xL,BCL-xS, BCL-xβ, BCL-x ΔTM and BCL-xy. Murine Bcl-x isoforms share a longN-terminal region (hatched).

FIG. 1C is a hydrophobicity plot of BCL-xL and BCL-xy.

FIG. 1D is an alignment of BCL-xy C-terminal sequence (residues 185–235of SEQ ID NO: 2) with an ankyrin-like consensus sequence (SEQ ID NO:26).

FIG. 2 shows SDS page analysis of protein products of BCL-xL, BCL-xS,BCL-xβ and BCL-xy after in vitro transcription and translation.

FIG. 3A shows expression of BCL-xy in different murine tissues by RT-PCR

FIG. 3B shows expression of BCL-x γ in lymph nodes of normal, nu/nu andRag 2^(−/−) mice.

FIG. 3C shows expression of BCL-x γ in the thymuses of normal,cortisone-treated and mutant mice as detected by RT-PCR.

FIG. 4A shows expression of BCL-x isoforms in activated T-cells afterCD3 ligation.

FIG. 4B shows expression of BCL-x isoforms in activated T-cells afterinterleukin-2 stimulation.

FIG. 5 shows apoptosis of BCL-x γ transfectants.

FIG. 6 shows expression of BCL-x isoforms in O3 T cell clone stimulatedby plate-bound anti-CD3 antibody for 5 hours and sorted by flowcytometry.

FIG. 7 shows expression of BCL-xγ in Balb/c thymocytes and DBA/2thymocytes as detected by RT-PCR.

FIG. 8 shows expression of BCL-xγ in CD69+ and CD69− thymocytes asdetected by RT-PCR

FIG. 9 shows expression of BCL-xy in thymocyte subpopulations.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to BCL-xy nucleic acid molecules,proteins, antibodies immunoreactive with BCL-xy proteins, andpreparations of such compositions. In addition, drug discovery assaysare provided for identifying other agents which can modulate thebiological function of BCL-xy proteins. Such agents are useful inmodulating growth, differentiation, and survival in a cell. As describedherein, BCL-xy modulating agents may be, inter alia, small organicmolecules, peptides or peptidomimetics, lipids, carbohydrates, ornucleic acids. Moreover, the present invention provides diagnostic andtherapeutic assays and reagents for detecting and treating disordersinvolving, for example, aberrant expression of mammalian bcl-xγ genes.Other aspects of the invention are described below or will be apparentto those skilled in the art in light of the present disclosure.

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode BCL-xγ or biologically active portions thereof, as well asnucleic acid fragments sufficient for use as hybridization probes toidentify BCL-xγ-encoding nucleic acid (e.g., BCL-xγ mRNA). As usedherein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA).The nucleic acid molecule may be single-stranded or double-stranded, butpreferably is double-stranded DNA. An “isolated” nucleic acid moleculeis free of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated BCL-xγ nucleic acidmolecule may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of nucleotide sequences which naturally flank the nucleicacid molecule in genomic DNA of the cell from which the nucleic acid isderived (e.g., a human splenocyte). Moreover, an “isolated” nucleic acidmolecule, such as a cDNA molecule, may be free of other cellularmaterial.

In one embodiment an isolated BCL-xy nucleic acid molecule of theinvention is a naturally occurring molecule. In a preferred embodiment,an isolated nucleic acid molecule of the invention comprises thenucleotide sequence shown in SEQ ID NO: 1. The sequence of SEQ ID NO: 1corresponds to the mouse BCL-xγ cDNA. This cDNAs comprises sequencesencoding the BCL-xγ protein (i.e., “the coding region”, from nucleotides378–1085 of SEQ ID NO:1), as well as 5′ untranslated sequences(nucleotides 1 to 377 of SEQ ID NO:1) and 3′ untranslated sequences(nucleotides 1083–1384 of SEQ ID NO:1). Alternatively, the nucleic acidmolecule may comprise only the coding region of SEQ ID NO: 1 (e.g.,nucleotides 378–1085).

The naturally occurring murine cDNA, comprises unique 3′ sequences. Theobserved sequence variations of the BCL-xy 3′ noncoding region representthe effects of insertions in two locations. The inserted fragments are,fragment B, represented by SEQ ID NO:3; fragment C, represented bynucleotides 1085–1193 of SEQ ID NO:1; fragment D represented by SEQ IDNO:4; and fragment E represented by nucleotides 1194–1384 of SEQ IDNO:1. Four types of 3′ UT variants have been defined as follows: (1)A-E; (2) A-C-E; (3) A-B-C-E; and (4) A-C-D-E.

Since all of the sequence variations are located in the 3′ noncodingregion, these variations do not represent potential artifact products ofPCR amplification. Since the length and content of the 3′ noncodingregion may affect mRNA translational efficiency or stability (Tanguayand Gallie, 1996 Mol. Cell. Biol. 16, 146–156), it will, in certainembodiments, be desirable to include portions of the 3′ noncodingregion. In one embodiment, a BCL-xγ nucleic acid molecule contains allor a portion of the 3′ untranslated region of SEQ ID NO:1, e.g.,nucleotides 1083–1384. In one embodiment, a BCL-xγ nucleic acid moleculecontains a sequence at least about 85% homologous to the sequence shownin nucleotides 1083–1384 SEQ ID NO:1. In another embodiment, a BCL-xγnucleic acid molecule contains a sequence at least about 90% homologousto the sequence shown in nucleotides 1083–1384 SEQ ID NO:1. In apreferred embodiment, a BCL-xγ nucleic acid molecule contains a sequenceat least about 95% homologous to the sequence shown in nucleotides1083–1384 SEQ ID NO:1.

In certain embodiments, the subject nucleic acid molecule includes oneor more of: fragment B, represented by SEQ ID NO:3; fragment C,represented by nucleotides 1085–1193 of SEQ ID NO:1; fragment Drepresented by SEQ ID NO:4; and fragment E represented by nucleotides1194–1384 of SEQ ID NO:1. In other embodiments the subject nucleic acidincludes the ordered combination of 3′ domains selected from the groupconsisting of: -E, -C-E, -B-C-E, and -C-D-E after the BCL-xγ stop codon.

Transcriptional regulatory sequences can control tissue specificexpression of genes. “Transcriptional regulatory sequence” is a termused throughout the specification to refer to DNA sequences, such asinitiation signals, enhancers, and promoters, which induce or controltranscription of protein coding sequences with which they areoperatively linked. In preferred embodiments, transcription of a bcl-xγgene is under the control of a promoter sequence (or othertranscriptional regulatory sequence) which controls the expression ofthe recombinant gene in a cell-type in which expression is intended. Itwill also be understood that the recombinant gene can be under thecontrol of transcriptional regulatory sequences which are the same orwhich are different from those sequences which control transcription ofthe naturally-occurring forms of BCL-xγ proteins.

In one embodiment a BCL-xγ nucleic acid further contains nucleotides1–164, i.e., the 5′ untranslated region of SEQ ID NO:1. In aparticularly preferred embodiment, a bcl-xγ gene is under the control ofa transcriptional regulatory sequence which includes nucleotides 1–164of SEQ ID NO:1. In another embodiment, a BCL-xγ nucleic acid contains anucleotide sequence at least about 80% homologous to the sequence shownin nucleotides 1–164. In another embodiment, a BCL-xγ nucleic acidcontains a nucleotide sequence at least about 85% homologous to thesequence shown in nucleotides 1–164. In a preferred embodiment, a BCL-xγnucleic acid contains a nucleotide sequence at least about 90%homologous to the sequence shown in nucleotides 1–164. In a particularlypreferred embodiment, a BCL-xγ nucleic acid contains a nucleotidesequence at least about 95% homologous to the sequence shown innucleotides 1–164.

This transcriptional regulatory sequence can also be used as part of atissue specific promoter to control the transcription of non-bcl-xγgenes, i.e., heterologous genes. As used herein, the term“tissue-specific promoter” means a nucleotide sequence that serves as apromoter, i.e., regulates expression of a selected nucleotide sequenceoperatively linked to the promoter, and which effects expression of theselected nucleotide sequence in specific cells of a tissue, such ascells of hepatic or pancreatic origin, neuronal cells, or immune cells.The term also covers so-called “leaky” promoters, which regulateexpression of a selected nucleic acid primarily in one tissue, but causeexpression in other tissues as well. Thus, in one embodiment of theinvention a transcriptional regulatory sequence including nucleotides1–164 of SEQ ID NO:1 is operatively linked to a heterologous codingsequence.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the coding region of SEQ ID NO: 1, for example a fragmentencoding a biologically active portion of BCL-xγ. The term “biologicallyactive portion of BCL-xγ” is intended to include portions of BCL-xγ thatretain anti-apoptotic activity. The ability of a portion of BCL-xγ tomodulate apoptosis can be determined in a number of assays, for example,by measuring the ability of a portion of bcl-x γ to modulate apoptosisafter T cell receptor ligation (described further in Example 9). Nucleicacid fragments encoding biologically active portions of BCL-xγ can beprepared by isolating a portion of SEQ ID NO: 1, expressing the encodedportion of BCL-xγ protein or peptide (e.g., by recombinant expression invitro) and assessing the anti-apoptotic activity of the encoded portionof BCL-xγ protein or peptide.

The BCL-xγ nucleic acid molecule shown in SEQ ID NO:1 was isolated froma mouse thymus cell cDNA library as described in Example 1. Othernaturally occurring BCL-xγ nucleic acid molecules, or portions thereof,can be isolated using standard molecular biology techniques and thesequence information provided herein. For example, a human BCL-xγ cDNAcan be isolated from a T cell line cDNA library using all or portion ofSEQ ID NO: 1 as a hybridization probe and standard hybridizationtechniques (e.g., as described in Sambrook, J., Fritsh, E. F., andManiatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). Moreover, anucleic acid molecule encompassing all or a portion of a nucleic acidmolecule homologous to SEQ ID NO: 1 can be isolated by the polymerasechain reaction using oligonucleotide primers designed based upon thesequence of SEQ ID NO: 1. For example, mRNA can be isolated from normalT cells (e.g., by the guanidinium-thiocyanate extraction procedure ofChirgwin et al. (1979) Biochemistry 18: 5294–5299) and cDNA can beprepared using reverse transcriptase (e.g., Moloney MLV reversetranscriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reversetranscriptase, available from Seikagaku America, Inc., St. Petersburg,Fla.). Synthetic oligonucleotide primers for PCR amplification can bedesigned based upon the nucleotide sequence shown in SEQ ID NO: 1. Forexample, primers suitable for amplification of the segment of SEQ ID NO:1 are shown in SEQ ID NOs: 6 and 7. A nucleic acid of the invention canbe amplified using cDNA or, alternatively, genomic DNA, as a templateand appropriate oligonucleotide primers according to standard PCRamplification techniques. The nucleic acid so amplified can be clonedinto an appropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to a BCL-xγ nucleotidesequence can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

Preferred BCL-xγ nucleic acid molecules are naturally-occurring nucleicacid molecules. As used herein, a “naturally-occurring” nucleic acidmolecule refers to an RNA or DNA molecule having a nucleotide sequencethat occurs in nature (e.g., encodes a natural protein). Such, nucleicacid molecules encoding BCL-xγ proteins from other species, and thuswhich have a nucleotide sequence which differs from the murine sequenceof SEQ ID NO: 1, are intended to be within the scope of the invention.In a preferred embodiment, the BCL-xγ nucleic acid molecule of thepresent invention is isolated from a vertebrate organism. More preferredBCL-xγ nucleic acids are mammalian. Particularly preferred BCL-xγnucleic acids are human or mouse in origin. In on embodiment, thenucleic acid encodes a natural murine BCL-xy. In a preferred embodiment,a BCL-xγ nucleic acid encodes the protein shown in SEQ ID NO: 2. Inanother embodiment, the nucleic acid molecule encodes a human homologueof murine BCL-xγ.

In addition to the BCL-xγ nucleotide sequence shown in SEQ ID NO: 1, itwill be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequences of BCL-xymay exist within a population (e.g., the human population). Such geneticpolymorphism in the bcl-xγ gene may exist among individuals within apopulation due to natural allelic variation. Any and all such nucleotidevariations and resulting amino acid polymorphisms in BCL-xγ that are theresult of natural allelic variation and that do not alter the functionalactivity of BCL-xγ are intended to be within the scope of the invention.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the BCL-xγ cDNAs of the invention can be isolated based ontheir homology to the BCL-xγ nucleic acid disclosed herein using thehuman cDNA, or a portion thereof, as a hybridization probe according tostandard hybridization techniques under stringent hybridizationconditions. As used herein, the term “hybridizes under stringentconditions” is intended to describe conditions for hybridization andwashing under which nucleotide sequences at least 60% homologous to eachother typically remain hybridized to each other. Preferably, theconditions are such that at least sequences at least 65%, morepreferably at least 70%, and even more preferably at least 75%homologous to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1–6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 50% formamide in 6× sodiumchloride/sodium citrate (SSC) at about 42° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50° C. then at 65° C. Preferred probes ofthe invention are those that hybridize under stringent conditions to thesequence shown in nucleotides 930–1082 of SEQ ID NO: 1. Accordingly, inanother embodiment, an isolated nucleic acid molecule of the inventionis at least 20 nucleotides in length and hybridizes under stringentconditions to the nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO: 1, preferably to a portion of the sequence shownin nucleotides 930–1082 of SEQ ID NO:1. In other embodiments, thenucleic acid is at least 30, 50, 100, 250, or 500 nucleotides in length.In preferred embodiments, the probe further contains a label group andcan be detected, e.g. the label group can be a radioisotope, fluorescentcompound, enzyme, or enzyme co-factor. Probes based on the subjectBCL-xγ sequences can also be used to detect transcripts or genomicsequences encoding the same or homologous proteins.

In addition to naturally-occurring allelic variants of the bcl-xγsequence that may exist in the population, the skilled artisan willfurther appreciate that changes may be introduced by mutation into thenucleotide sequence of SEQ ID NO: 1, thereby leading to changes in theamino acid sequence of the encoded BCL-xγ protein, without altering thefunctional ability of the BCL-xγ protein. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues may be made in the sequence of SEQ ID NO: 1. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of BCL-xγ (e.g., the sequence of SEQ ID NO: 2)without altering the anti-apoptotic activity of BCL-xγ, whereas an“essential” amino acid residue is required for BCL-xγ anti-apoptoticactivity, e.g., in a thymic cell. Amino acid residues of BCL-xγ that arestrongly conserved among members of the BCL family are predicted to beessential in BCL-xγ and thus are not likely to be amenable tosignificant alteration.

For example, the BCL-xγ proteins of the present invention contain theBH1–4 domains conserved in BCL-2-related proteins (D'Sa-Eipper et al.1996. Cancer Res. 56:3879). Since these domains are conserved among theBCL proteins, they may be less amenable to alteration. In addition, theBCL-xγ proteins of the present invention contain several otherstructural features, or domains. For example, the γ domain is shown inamino acids 185–235 of SEQ ID NO:2. BCL-x γ is the first proteindemonstrated to contain a γ domain sequence. The γ domain does not sharea high degree of homology with any known protein. The γ domain does,however, contain a 33 amino acid portion that shows strong homology withthe consensus sequence for ankyrin-like domains (Hatada et al., 1992Proc. Natl. Acad. Sci. USA 89, 2489–2493). Since this unique domain islikely responsible for the unique role of BCL-xγ in apoptosis and thymicdevelopment, it may be less amenable to manipulation.

In one embodiment a BCL-xγ nucleic acid molecule comprises a nucleotidesequence at least about 80% homologous to nucleotides 930–1082 of SEQ IDNO:1, which encode a γ domain. In a preferred embodiment, a BCL-xγnucleic acid contains a sequence at least about 90% homologous tonucleotides 930–1082 of SEQ ID NO:1. In another preferred embodiment, aBCL-xγ nucleic acid of the present invention contains a nucleotidesequence at least about 95% homologous to nucleotides 930–1082 of SEQ IDNO:1. In a particularly preferred embodiment, a BCL-xγ nucleic acidcontains a nucleotide sequence shown in nucleotides 930–1082 of SEQ IDNO:1.

In another embodiment a BCL-xγ nucleic acid molecule encodes a proteinwhich comprises a sequence at least about 60% homologous to a γ domainshown in amino acids 185–235 of SEQ ID NO:2 and having anti-apoptoticactivity. In a preferred embodiment a BCL-xγ nucleic acid moleculeencodes a protein which comprises a sequence at least about 70%homologous to a γ domain shown in amino acids 185–235 of SEQ ID NO:2 andhaving anti-apoptotic activity. In another preferred embodiment a BCL-xγnucleic acid molecule encodes a protein which comprises a sequence atleast about 80% homologous to a γ domain shown in amino acids 185–235 ofSEQ ID NO:2 and having anti-apoptotic activity. In yet another preferredembodiment a BCL-xγ nucleic acid molecule encodes a protein whichcomprises a sequence at least about 90% homologous to a γ domain shownin amino acids 185–235 of SEQ ID NO:2 and having anti-apoptoticactivity. In another preferred embodiment a BCL-xγ nucleic acid moleculeencodes a protein which comprises a sequence at least about 95%homologous to a γ domain shown in amino acids 185–235 of SEQ ID NO:2 andhaving anti-apoptotic activity. In a particularly preferred embodiment aBCL-xγ nucleic acid molecule encodes a protein which comprises asequence shown in amino acids 185–235 of SEQ ID NO:2.

The BCL-xγ protein also has an ankyrin domain. Ankyrin domains define avariety of proteins, including the protooncogene Bcl-3 (Ohno et al.,1990 Cell 60:991), that may use this sequence to bind NF-κB p50 andregulate the activation of this transcription factor ((Hatada et al.,1992 Proc. Natl. Acad. Sci. USA 89, 2489–2493)). In one embodiment aBCL-xγ nucleic acid molecule encodes an intracellular protein with aconsensus ankyrin domain shown in the sequence NXXXXXXGXTPLXX (SEQ IDNO: 25), which is anti-apoptotic. In a preferred embodiment a BCL-xγnucleic acid molecule encodes an intracellular protein with the ankyrindomain shown in amino acids 185–217 of SEQ ID NO:2 which isanti-apoptotic.

Another aspect of the invention pertains to nucleic acid moleculesencoding BCL-xγ proteins that contain changes in amino acid residuesthat are not essential for anti-apoptotic activity. Such BCL-xγ proteinsdiffer in amino acid sequence from SEQ ID NO: 2 yet retainanti-apoptotic activity. In one embodiment, the isolated nucleic acidmolecule comprises a nucleotide sequence encoding a protein, wherein theprotein comprises an amino acid sequence at least 83.5% homologous tothe amino acid sequence of SEQ ID NO: 2 and having an anti-apoptoticactivity. Preferably, the protein encoded by the nucleic acid moleculeis at least 84% homologous to SEQ ID NO: 2, more preferably at least 85%homologous to SEQ ID NO: 2, even more preferably at least 90% homologousto SEQ ID NO: 2 and has anti-apoptotic activity. In a preferredembodiment the protein encoded by the nucleic acid molecule is at least95% homologous to the amino acid sequence shown in SEQ ID NO:2. Apreferred sequence is identical to the sequence shown in SEQ ID NO:2 andhas anti-apoptotic activity.

To determine the percent homology of two amino acid sequences (e.g., SEQID NO: 2 and a mutant form thereof), the sequences are aligned foroptimal comparison purposes (e.g., gaps may be introduced in thesequence of one protein for optimal alignment with the other protein).The amino acid residues at corresponding amino acid positions are thencompared. When a position in one sequence (e.g., SEQ ID NO: 2) isoccupied by the same or a similar amino acid residue as thecorresponding position in the other sequence (e.g., a mutant form ofBCL-xγ), then the molecules are homologous at that position (i.e., asused herein amino acid “homology” is equivalent to amino acid identityor similarity). As used herein, an amino acid residue is “similar” toanother amino acid residue if the two amino acid residues are members ofthe same family of residues having similar side chains. Families ofamino acid residues having similar side chains have been defined in theart, including basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). The percent homology between two sequences, therefore, is afunction of the number of identical or similar positions shared by twosequences (i.e., % homology=# of identical or similar positions/total #of positions×100).

In a preferred embodiment a BCL-xγ nucleic acid is at least about 92%,93%, or 94% homologous to the coding sequence shown in SEQ ID NO:1 orits complement. In another preferred embodiment a BCL-xγ nucleic acid isat least about 95% homologous to the coding sequence shown in SEQ IDNO:1 or its complement. In yet another preferred embodiment a BCL-xγnucleic acid is at least about 97–98% homologous to the coding sequenceshown in SEQ ID NO:1. In a preferred embodiment, a BCL-xγ nucleic acidcomprises a nucleotide sequence which is identical to the codingsequence of SEQ ID NO: 1.

An isolated nucleic acid molecule encoding a BCL-xγ protein homologousto the protein of SEQ ID NO: 2 can be created by introducing one or morenucleotide substitutions, additions or deletions into the nucleotidesequence of SEQ ID NO: 1 such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.Mutations can be introduced into SEQ ID NO: 1 by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore predicted non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart, and are defined above. Thus, a predicted nonessential amino acidresidue in BCL-xγ is preferably replaced with another amino acid residuefrom the same side chain family. Alternatively, in another embodiment,mutations can be introduced randomly along all or part of a BCL-xγcoding sequence, such as by saturation mutagenesis, and the resultantmutants can be screened for proteolytic activity to identify mutantsthat retain proteolytic activity. Following mutagenesis of SEQ ID NO: 1,the encoded protein can be expressed recombinantly (e.g., bytransfection as described in Example 9) and the anti-apoptotic activityof the protein can be determined.

A suitable assay for testing the anti-apoptotic activity of portions ofBCL-xγ proteins and mutated BCL-xγ proteins is described in detail inExample 9. Briefly, the percentage of cells undergoing apoptosis after Tcell receptor ligation can be analyzed using propidium iodide staining.Numerous other methods for measuring apoptosis are known in the art andmany assays are commercially available, such as, the DNA fragmentationELISA, the TUNEL assay, and the apoptotic DNA ladder kit, all fromBoehringer Mannheim.

A. Sources of Nucleic Acids

Bcl-xγ nucleic acids can be obtained from mRNA present in any of anumber of eukaryotic cells. It should also be possible to obtain nucleicacids encoding mammalian BCL-xγ proteins of the present invention fromgenomic DNA from both adults and embryos. For example, a gene encoding aBCL-xγ protein can be cloned from either a cDNA or a genomic library inaccordance with protocols described herein, as well as those generallyknown to persons skilled in the art. Examples of tissues and/orlibraries suitable for isolation of the subject nucleic acids include Tcells, among others. A cDNA encoding a BCL-xγ protein can be obtained byisolating total mRNA from a cell, e.g. a vertebrate cell, a mammaliancell, or a human cell, including embryonic cells. Double stranded cDNAscan then be prepared from the total mRNA, and subsequently inserted intoa suitable plasmid or bacteriophage vector using any one of a number ofknown techniques. The gene encoding a mammalian BCL-xγ protein can alsobe cloned using established polymerase chain reaction techniques inaccordance with the nucleotide sequence information provided by theinvention. The nucleic acid of the invention can be DNA or RNA. Apreferred nucleic acid is a cDNA represented by a sequence shown in SEQID NO: 1.

Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding the antisense RNA molecule. SuchDNA sequences may be incorporated into a wide variety of vectors whichincorporate suitable RNA polymerase promoters such as the T7 or SP6polymerase promoters. Alternatively, antisense cDNA constructs thatsynthesize antisense RNA constitutively or inducibly, depending on thepromoter used, can be introduced stably into cell lines.

Any of the subject nucleic acids can also be obtained by chemicalsynthesis. For example, nucleic acids of the invention may besynthesized by standard methods known in the art, e.g. by use of anautomated DNA synthesizer (such as are commercially available fromBiosearch, Applied Biosystems, etc.). As examples, phosphorothioateoligonucleotides may be synthesized by the method of Stein et al. (1988.Nucl. Acids Res. 16:3209). methylphosphonate oligonucleotides can beprepared by use of controlled pore glass polymer supports (Sarin et al.,1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448–7451), etc. Other techniquesfor chemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis.

Moreover, various well-known modifications to nucleic acid molecules maybe introduced as a means of increasing intracellular stability andhalf-life. Possible modifications include but are not limited to theaddition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule or the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages within the oligodeoxyribonucleotide backbone.

The subject nucleic acids may also contain modified bases. For example,a nucleic acid may comprise at least one modified base moiety which isselected from the group including but not limited to 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine.

A modified nucleic acid of the present invention may also include atleast one modified sugar moiety selected from the group including butnot limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the subject nucleic acid may include at leastone modified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

B. Nucleic Acid Probes

In another embodiment a BCL-xγ nucleic acid fragment is anoligonucleotide probe which specifically detects a BCL-xγ nucleic acidrelative to another, related BCL nucleic acid. In a preferredembodiment, the subject oligonucleotide hybridizes under stringentconditions to a nucleic acid with at least about 6 consecutivenucleotides encoding a γ domain, for example, nucleotides 930–1082 ofSEQ ID NO:1.

In preferred embodiments, the probe further contains a label group andcan be detected, e.g. the label group can be a radioisotope, fluorescentcompound, enzyme, biotin, or enzyme co-factor. Probes based on thesubject BCL-xγ sequences can also be used to detect transcripts orgenomic sequences encoding the same or homologous proteins.

C. Antisense Constructs

Another aspect of the invention relates to the use of isolated BCL-xγnucleic acids in “antisense” therapy. As used herein, “antisense”therapy refers to administration or in situ generation ofoligonucleotide molecules or their derivatives which specificallyhybridize (e.g. bind) under cellular conditions, with the cellular mRNAand/or genomic DNA encoding one or more of the subject BCL-xγ proteinsso as to inhibit expression of that protein, e.g. by inhibitingtranscription and/or translation. The binding may be by conventionalbase pair complementarity, or, for example, in the case of binding toDNA duplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” therapy refers to the range oftechniques generally employed in the art, and includes any therapy whichrelies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thecellular mRNA which encodes a mammalian BCL-xγ protein. Alternatively,the antisense construct is an oligonucleotide probe which is generatedex vivo and which, when introduced into the cell causes inhibition ofexpression by hybridizing with the mRNA and/or genomic sequences of amammalian bcl-xγ gene. Such oligonucleotide probes are preferablymodified oligonucleotides which are resistant to endogenous nucleases,e.g. exonucleases and/or endonucleases, and are therefore stable invivo. Exemplary nucleic acid molecules for use as antisenseoligonucleotides are phosphoramidate, phosphothioate andmethylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996;5,264,564; and 5,256,775). Additionally, general approaches toconstructing oligomers useful in antisense therapy have been reviewed,for example, by Van der Krol et al. (1988) Biotechniques 6:958–976; andStein et al. (1988) Cancer Res 48:2659–2668.

Antisense approaches involve the design of oligonucleotides (either DNAor RNA) that are complementary to BCL-xγ mRNA. The antisenseoligonucleotides will bind to the BCL-xγ mRNA transcripts and preventtranslation. Absolute complementarity, although preferred, is notrequired. A sequence “complementary” to a portion of an RNA, as referredto herein, means a sequence having sufficient complementarity to be ableto hybridize with the RNA, forming a stable duplex. In the case ofdouble-stranded antisense nucleic acids, a single strand of the duplexDNA may thus be tested, or triplex formation may be assayed. The abilityto hybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches with an RNA it maycontain and still form a stable duplex (or triplex, as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, are preferred. However, sequences complementary to the3′ untranslated sequences of mRNAs may also be used (Wagner, R. 1994.Nature 372:333). Therefore, oligonucleotides complementary to either the5′ or 3′ untranslated, non-coding regions of a bcl-xγ gene can be usedin an antisense approach to inhibit translation of endogenous BCL-xγmRNA. Oligonucleotides complementary to the 5′ untranslated region ofthe mRNA preferably include the complement of the AUG start codon.Antisense oligonucleotides complementary to mRNA coding regions may alsobe used in accordance with the invention. Whether designed to hybridizeto the 5′, 3′ or coding region of BCL-xγ mRNA, antisense nucleic acidsshould be at least about six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Incertain embodiments, the oligonucleotide is at least about 10nucleotides, at least about 17 nucleotides, at least about 25nucleotides, or at least about 50 nucleotides.

Regardless of the choice of target sequence, in vitro studies can beperformed to quantitate the ability of the antisense oligonucleotide toinhibit gene expression. These studies can utilize controls thatdistinguish between antisense gene inhibition and nonspecific biologicaleffects of oligonucleotides. Levels of the target RNA or protein can becompared with that of an internal control RNA or protein. Resultsobtained using the antisense oligonucleotide can be compared with thoseobtained using a control oligonucleotide. The control oligonucleotidecan be approximately the same length as the test oligonucleotide andthat the nucleotide sequence of the oligonucleotide differs from theantisense sequence no more than is necessary to prevent specifichybridization to the target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as: peptides (e.g., for targeting host cellreceptors in vivo); or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553–6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci.84:648–652; PCT Publication No. W088/09810, published Dec. 15, 1988); orthe blood-brain barrier (see, e.g., PCT Publication No. W089/10134,published Apr. 25, 1988); hybridization-triggered cleavage agents; (See,e.g., Krol et al., 1988, BioTechniques 6:958–976); and/or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539–549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

While antisense nucleotides complementary to the bcl-xγ coding regionsequence can be used, those complementary to the transcribeduntranslated region are most preferred. Exemplary antisenseoligonucleotides are set forth in SEQ ID NOs. 19, 20, 21, or 22.

The antisense molecules can be delivered to cells which express theBCL-xγ in vivo or in vitro. A number of methods have been developed fordelivering antisense DNA or RNA to cells; e.g., antisense molecules canbe injected directly into the tissue site, or modified antisensemolecules designed to target the desired cells (e.g., antisense linkedto peptides or antibodies that specifically bind receptors or antigensexpressed on the target cell surface) can be administeredsystematically.

A preferred approach utilizes a recombinant DNA construct in which theantisense oligonucleotide is placed under the control of a strong polIII or pol II promoter. The use of such a construct to transfect targetcells in the patient will result in the transcription of sufficientamounts of single stranded RNAs that will form complementary base pairswith the endogenous BCL-xγ transcripts and thereby prevent translationof the BCL-xγ mRNA. For example, a vector can be introduced in vivo suchthat it is taken up by a cell and directs the transcription of anantisense RNA. Such a vector can remain episomal or become chromosomallyintegrated, as long as it can be transcribed to produce the desiredantisense RNA. Such vectors can be constructed by recombinant DNAtechnology methods standard in the art. Vectors can be plasmid, viral,or others known in the art, used for replication and expression inmammalian cells. Expression of the sequence encoding the antisense RNAcan be by any promoter known in the art to act in mammalian, preferablyhuman cells. Such promoters can be inducible or constitutive. Suchpromoters include but are not limited to: the SV40 early promoter region(Bernoist and Chambon, 1981, Nature 290:304–310), the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al.,1980, Cell 22:787–797), the herpes thymidine kinase promoter (Wagner etal., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441–1445), the regulatorysequences of the metallothionein gene (Brinster et al, 1982, Nature296:39–42), etc. Any type of plasmid, cosmid, yeast artificalchromosome, YAC, or viral vector can be used to prepare the recombinantDNA construct which can be introduced directly into the tissue site;e.g., the choroid plexus or hypothalamus. Alternatively, viral vectorscan be used which selectively infect the desired tissue (e.g., forbrain, herpesvirus vectors may be used), in which case administrationmay be accomplished by another route (e.g., systemically).

Ribozyme molecules designed to catalytically cleave BCL-xγ mRNAtranscripts can also be used to prevent translation of BCL-xγ mRNA andexpression of BCL-xγ. (See, e.g., PCT International PublicationWO90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science247:1222–1225). While ribozymes that cleave mRNA at site specificrecognition sequences can be used to destroy BCL-xγ mRNAs, the use ofhammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions that form complementary basepairs with the target mRNA. The sole requirement is that the target mRNAhave the following sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, 1988, Nature, 334:585–591.There are numerous potential hammerhead ribozyme cleavage sites withinthe nucleotide sequence of BCL-xγ cDNA. Preferably, the ribozyme isengineered so that the cleavage recognition site is located near the 5′end of the BCL-xγ specific mRNA; i.e., to increase efficiency andminimize the intracellular accumulation of non-functional mRNAtranscripts.

Ribozymes of the present invention also include RNA endoribonucleases(hereinafter “Cech-type ribozymes”) such as the one which occursnaturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA)and which has been extensively described by Thomas Cech andcollaborators (Zaug, et al., 1984, Science, 224:574–578; Zaug and Cech,1986, Science, 231:470–475; Zaug, et al., 1986, Nature, 324:429–433;published International patent application No. WO88/04300 by UniversityPatents Inc.; Been and Cech, 1986, Cell, 47:207–216). The Cech-typeribozymes have an eight base pair active site which hybridizes to atarget RNA sequence whereafter cleavage of the target RNA takes place.The invention encompasses those Cech-type ribozymes which target eightbase-pair active site sequences that are present in BCL-xγ mRNA.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g. for improved stability, targeting, etc.) andshould be delivered to cells which express the BCL-xγ in vivo e.g., Tcells or thymocytes. A preferred method of delivery involves using a DNAconstruct “encoding” the ribozyme under the control of a strongconstitutive promoter such as the pol III or pol II promoter, so thattransfected cells will produce sufficient quantities of the ribozyme todestroy endogenous BCL-xγ and inhibit translation. Bccause ribozymesunlike antisense molecules, are catalytic, a lower intracellularconcentration is required for efficiency.

Endogenous bcl-xγ gene expression can also be reduced by inactivating or“knocking out” the bcl-xγ gene or its promoter using targeted homologousrecombination. (e.g., see Smithies et al., 1985, Nature 317:230–234;Thomas & Capecchi, 1987, Cell 51:503–512; Thompson et al., 1989 Cell5:313–321; each of which is incorporated by reference herein in itsentirety). For example, a mutant, non-functional bcl-xγ (or a completelyunrelated DNA sequence) flanked by DNA homologous to the endogenousbcl-xγ gene (either the coding regions or regulatory regions of thebcl-xγ gene) can be used, with or without a selectable marker and/or anegative selectable marker, to transfect cells that express BCL-xγ invivo. Insertion of the DNA construct, via targeted homologousrecombination, results in inactivation of the bcl-xγ gene. Suchapproaches are particularly suited in the generation of animal offspringwith an inactive BCL-xγ (e.g., see Thomas & Capecchi 1987 and Thompson1989, supra). However this approach can be adapted for use in humansprovided appropriate delivery means are used.

Alternatively, endogenous bcl-xγ gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the bcl-xy gene (i.e., the bcl-xy promoter and/or enhancers)to form triple helical structures that prevent transcription of thebcl-xγ gene in target cells in the body. (See generally, Helene, C.1991, Anticancer Drug Des., 6(6):569–84; Helene, C., et al., 1992, Ann,N.Y. Accad. Sci., 660:27–36; and Maher, L. J., 1992, Bioassays14(12):807–15).

Nucleic acid molecules to be used in triple helix formation for theinhibition of transcription are preferably single stranded and composedof deoxyribonucleotides. The base composition of these oligonucleotidesshould promote triple helix formation via Hoogsteen base pairing rules,which generally require sizable stretches of either purines orpyrimidines to be present on one strand of a duplex. Nucleotidesequences may be pyrimidine-based, which will result in TAT and CGCtriplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementarity to apurine-rich. region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich. These molecules will form a triple helixwith a DNA duplex that is rich in GC pairs, in which the majority of thepurine residues are located on a single strand of the targeted duplex,resulting in CGC triplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′, 3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625–6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131–6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327–330). Exemplary modified bases are set forth above.

D. Modifications to Nucleic Acids

Modifications to nucleic acid molecules of the invention can beintroduced as a means of increasing intracellular stability andhalf-life. Modifications include but are not limited to the addition offlanking sequences of ribonucleotides or deoxyribonucleotides to the 5′and/or 3′ ends of the molecule or the use of phosphorothioate or 2′O-methyl rather than phosphodiesterase linkages within theoligodeoxyribonucleotide backbone. Modified bases are known in the artand are described above.

II. Expression Vectors and Host Cells

The present invention also provides for vectors containing the subjectnucleic acid molecules. As used herein, the term “vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of preferred vector is an episome,i.e., a nucleic acid capable of extra-chromosomal replication. Preferredvectors are those capable of autonomous replication and/expression ofnucleic acids to which they are linked. Vectors capable of directing theexpression of genes to which they are operatively linked are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of“plasmids” which refer generally to circular double stranded DNA loopswhich, in their vector form are not bound to the chromosome. In thepresent specification, “plasmid” and “vector” are used interchangeablyas the plasmid is the most commonly used form of vector. However, theinvention is intended to include such other forms of expression vectorswhich serve equivalent functions.

This invention also provides expression vectors containing a nucleicacid encoding a BCL-xγ protein, operatively linked to at least onetranscriptional regulatory sequence. “Operatively linked” is intended tomean that the nucleotide sequence is linked to a regulatory sequence ina manner which allows expression of the nucleotide sequence.Transcriptional regulatory sequences are art-recognized and are selectedto direct expression of the subject mammalian BCL-xγ proteins. Exemplaryregulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990).

In a preferred embodiment the expression vector of the present inventionis capable of replicating in a cell. In one embodiment, the expressionvector includes a recombinant gene encoding a peptide having BCL-xγanti-apoptotic activity. Such expression vectors can be used totransfect cells and thereby produce proteins, including fusion proteins,encoded by nucleic acids as described herein. Moreover, the geneconstructs of the present invention can also be used as a part of a genetherapy protocol to deliver nucleic acids encoding either an agonisticor antagonistic form of one of the subject mammalian BCL-xγ proteins.Thus, another aspect of the invention features expression vectors for invivo or in vitro transfection and expression of a mammalian BCL-xγprotein in particular cell types so as to reconstitute the function of,or alternatively, abrogate the function of BCL-xγ in a tissue. Thiscould be desirable when treating a disorder, for example, resulting fromthe misexpression of BCL-xγ in a tissue.

In addition to viral transfer methods, such as those described above,non-viral methods can also be employed to cause expression of a subjectBCL-xγ protein in the tissue of an animal. Most nonviral methods of genetransfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral targeting means of the present invention rely onendocytic pathways for the uptake of the subject BCL-xγ protein gene bythe targeted cell. Exemplary targeting means of this type includeliposomal derived systems, poly-lysine conjugates, and artificial viralenvelopes.

The recombinant bcl-xγ genes can be produced by ligating nucleic acidencoding a BCL-xγ protein, or a portion thereof, into a vector suitablefor expression in either prokaryotic cells, eukaryotic cells, or both.Expression vectors for production of recombinant forms of the subjectBCL-xγ proteins include plasmids and other vectors. For instance,suitable vectors for the expression of a BCL-xγ protein include plasmidsof the types: pBR322-derived plasmids, pEMBL-derived plasmids,pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmidsfor expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al. (1983) inExperimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, a BCL-xγ protein is producedrecombinantly utilizing an expression vector generated by sub-cloningthe coding sequence of one of the bcl-xγ genes represented in SEQ IDNO:1.

The preferred mammalian expression vectors contain both prokaryoticsequences, to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papillomavirus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989) Chapters 16 and 17.

In some instances, it may be desirable to express the recombinant BCL-xγprotein by the use of a baculovirus expression system. Examples of suchbaculovirus expression systems include pVL-derived vectors (such aspVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1),and pBlueBac-derived vectors (such as the β-gal containing pBlueBacIII).

In some cases it will be desirable to express only a portion of a BCL-xγprotein. The subject vectors can also include fragments of a BCL-xγnucleic acid encoding a fragment of a BCL-xγ protein, preferably afragment having anti-apoptotic activity.

The subject vectors can be used to transfect a host cell in order toexpress a recombinant form of the subject BCL-xγ proteins. The host cellmay be any prokaryotic or eukaryotic cell. Thus, a nucleotide sequencederived from the cloning of mammalian BCL-xγ proteins, encoding all or aselected portion of the full-length protein, can be used to produce arecombinant form of a mammalian BCL-xγ protein in a cell.

“Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

The present invention further pertains to methods of producing thesubject BCL-xγ proteins. For example, a host cell transfected with anucleic acid vector directing expression of a nucleotide sequenceencoding the subject proteins can be cultured under appropriateconditions to allow expression of the peptide to occur. The cells may beharvested, lysed and the protein isolated. A cell culture includes hostcells, media and other byproducts. Suitable media for cell culture arewell known in the art. The recombinant BCL-xγ protein can be isolatedfrom cell culture medium, host cells, or both using techniques known inthe art for purifying proteins including ion-exchange chromatography,gel filtration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for such peptide.In a preferred embodiment, the recombinant BCL-xγ protein is a fusionprotein containing a domain which facilitates its purification, such asGST fusion protein or poly(His) fusion protein.

In other embodiments transgenic animals, described in more detail belowcan be used to produce recombinant proteins.

The present invention also provides for a recombinant transfectionsystem, including a bcl-xγ gene construct operatively linked to atranscriptional regulatory sequence and a gene delivery composition fordelivering the gene construct to a cell so that the cell expresses theBCL-xγ protein.

As used herein, the term “transfection” means the introduction of anucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of a mammalian BCL-xγprotein or, in the case of anti-sense expression from the transferredgene, the expression of a naturally-occurring form of the BCL-xγ proteinis disrupted.

A “delivery composition” shall mean a targeting means (e.g. a moleculethat results in higher affinity binding of a gene, protein, protein orpeptide to a target cell surface and/or increased cellular uptake by atarget cell). Examples of targeting means include: sterols (e.g.cholesterol), lipids (e.g. a cationic lipid, virosome or liposome),viruses (e.g. adenovirus, adeno-associated virus, and retrovirus) ortarget cell specific binding agents (e.g. ligands recognized by targetcell specific receptors).

III. Proteins

The present invention further pertains to isolated and/or recombinantforms of a BCL-xγ protein.

The term “recombinant protein” refers to a protein of the presentinvention which is produced by recombinant DNA techniques, whereingenerally, DNA encoding a mammalian BCL-xγ protein is inserted into asuitable expression vector which is in turn used to transform a hostcell to produce the heterologous protein, as described above. Moreover,the phrase “derived from”, with respect to a recombinant bcl-xγ gene, ismeant to include within the meaning of “recombinant protein” thoseproteins having an amino acid sequence of a natural occurring BCL-xγprotein, or a similar amino acid sequence which is generated bymutations including substitutions and deletions (including truncation)of a naturally occurring form of the protein.

The present invention also makes available isolated BCL-xγ proteinswhich are isolated from, or otherwise substantially free from othercellular proteins, especially other factors which may normally beassociated with the BCL-xγ protein. The term “substantially free ofother cellular proteins” (also referred to herein as “contaminatingproteins”) or “substantially pure or purified preparations” are definedas encompassing preparations of BCL-xγ proteins having less than about20% (by dry weight) contaminating protein, and preferably having lessthan about 5% contaminating protein. Functional forms of the subjectproteins can be prepared, for the first time, as purified preparationsby using a cloned gene as described herein. By “purified”, it is meant,when referring to a peptide or DNA or RNA sequence, that the indicatedmolecule is present in the substantial absence of other biologicalmacromolecules, such as other proteins. The term “purified” as usedherein preferably means at least about 80% by dry weight, morepreferably in the range of 95–99% by weight, and most preferably atleast about 99.8% by weight, of biological macromolecules of the sametype present (but water, buffers, and other small molecules, especiallymolecules having a molecular weight of less than 5000, can be present).The term “pure” as used herein preferably has the same numerical limitsas “purified” immediately above. “Isolated” and “purified” are not meantto encompass either natural materials in their native state or naturalmaterials that have been separated into components (e.g., in anacrylamide gel) but not obtained either as pure (e.g. lackingcontaminating proteins, or chromatography reagents such as denaturingagents and polymers, e.g. acrylamide or agarose) substances orsolutions. In preferred embodiments, purified BCL-xγ preparations willlack any contaminating proteins from the same animal from which BCL-xγis normally produced, as can be accomplished by recombinant expressionof, for example, a human BCL-xγ protein in a non-human cell.

In a preferred embodiment a BCL-xγ protein includes the amino acidsequence shown in SEQ ID NO:2. In other embodiments, a BCL-xγ protein iscapable of modulating apoptosis in a T cell.

The present invention also provides for BCL-xγ proteins which have aminoacid sequences evolutionarily related to the BCL-xγ proteins representedin SEQ ID NO: 2. In a preferred embodiment, a BCL-xγ protein of thepresent invention is a mammalian BCL-xγ protein. The term“evolutionarily related to”, with respect to amino acid sequences ofmammalian BCL-xγ proteins, refers to both proteins having amino acidsequences which have arisen naturally, and also to mutational variantsof mammalian BCL-xγ proteins which are derived, for example, bycombinatorial mutagenesis. Such related BCL-xγ proteins preferred by thepresent invention are at least about 83.5% homologous with the aminoacid sequence shown in SEQ ID NO: 2. In other embodiments, a BCL-xγprotein is at least about 85% homologous with the amino acid sequenceshown in SEQ ID NO: 2. In a preferred embodiment, a BCL-xγ protein is atleast about 90% homologous with the amino acid sequence shown in SEQ IDNO: 2. In another preferred embodiment, a BCL-xγ protein is at leastabout 95% homologous with the amino acid sequence shown in SEQ ID NO: 2.

In certain embodiments, it will be advantageous to alter a BCL-xγprotein to provide homologs of one of the subject BCL-xγ proteins whichwould function in some capacity as either a BCL-xγ agonist (mimetic) ora BCL-xγ antagonist, in order to promote or inhibit only a subset of thebiological activities of the naturally-occurring form of the protein.Thus, specific biological effects can be elicited by treatment with ahomolog of limited function, and with fewer side effects relative totreatment with agonists or antagonists which are directed to all of thebiological activities of naturally occurring forms of BCL-xγ proteins.

Homologs of each of the subject BCL-xγ proteins can be generated bymutagenesis, such as by discrete point mutation(s), or by truncation.For instance, mutation can give rise to homologs which retainsubstantially the same, or merely a subset, of the biological activityof the BCL-xγ protein from which it was derived. Alternatively,antagonistic forms of the protein can be generated which are able toinhibit the function of the naturally occurring form of the protein,such as by competitively binding to a BCL-xγ binding protein. Inaddition, agonistic forms of the protein may be generated which areconstitutively active. Thus, the mammalian BCL-xγ protein and homologsthereof provided by the subject invention may be either positive ornegative regulators of apoptosis.

The recombinant BCL-xγ proteins of the present invention includehomologs of the wild type BCL-xγ proteins, such as versions of thoseprotein which are resistant to proteolytic cleavage, as for example, dueto mutations which alter ubiquitination or other enzymatic targetingassociated with the protein. The subject proteins can also beglycosylated. A “glycosylated” BCL-xγ protein is an BCL-xγ proteinhaving a covalent linkage with a glycosyl group (e.g. a derivatized witha carbohydrate). An unglycosylated BCL-xγ protein can be generated byexpression in a system which is defective for glycosylation, such as abacterial cell. Alternatively, an existing glycosylation site can bemutated to preclude carbohydrate attachment. Likewise, new glycosylationsites, such as for N-linked or O-linked glycosylation, can be added byrecombinant techniques.

BCL-xγ proteins may also be chemically modified to create BCL-xγderivatives by forming covalent or aggregate conjugates with otherchemical moieties, such as lipids, phosphate, acetyl groups and thelike. Covalent derivatives of BCL-xγ proteins can be prepared by linkingthe chemical moieties to functional groups on amino acid sidechains ofthe protein or at the N-terminus or at the C-terminus of the protein.

Modification of the structure of the subject mammalian BCL-xγ proteinscan be for such purposes as enhancing therapeutic or prophylacticefficacy, stability (e.g., ex vivo shelf life and resistance toproteolytic degradation in vivo), or post-translational modifications(e.g., to alter phosphorylation pattern of the protein). Such modifiedpeptides, when designed to retain at least one activity of thenaturally-occurring form of the protein, or to produce specificantagonists thereof, are considered functional equivalents of the BCL-xγproteins described in more detail herein. Such modified peptides can beproduced, for instance, by amino acid substitution, deletion, oraddition.

For example, it is reasonable to expect that an isolated replacement ofa leucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid (i.e. isosteric and/or isoelectricmutations) will not have a major effect on the biological activity ofthe resulting molecule. Conservative replacements are those that takeplace within a family of amino acids that are related in their sidechains. Genetically encoded amino acids can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. In similarfashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine andmethionine. (see, for example, Biochemistry, 2nd ed., Ed. by L. Stryer,W H Freeman and Co.: 1981). Whether a change in the amino acid sequenceof a peptide results in a functional BCL-xγ homolog (e.g. functional inthe sense that the resulting protein mimics or antagonizes the wild-typeform) can be readily determined by assessing the ability of the variantpeptide to produce a response in cells in a fashion similar to thewild-type protein as discussed herein, or competitively inhibit such aresponse. Proteins in which more than one replacement has taken placecan readily be tested in the same manner.

In another embodiment, a BCL-xγ protein is encoded by a BCL-xγ nucleicacid as defined herein.

Full length proteins or fragments corresponding to one or moreparticular motifs and/or domains or to arbitrary sizes, for example, atleast about 5, 10, 25, 50, 75, 100, 125, 150 amino acids in length arewithin the scope of the present invention. For example, isolated BCL-xγproteins can include all or a portion of an amino acid sequencecorresponding to a BCL-xγ protein represented in or homologous to SEQ IDNO:2. Isolated peptidyl portions of BCL-xγ proteins can be obtained byscreening peptides recombinantly produced from the correspondingfragment of the nucleic acid encoding such peptides. In addition,fragments can be chemically synthesized using techniques known in theart such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, a BCL-xγ protein of the present invention may bearbitrarily divided into fragments of desired length with no overlap ofthe fragments, or preferably divided into overlapping fragments of adesired length. The fragments can be produced (recombinantly or bychemical synthesis) and tested to identify those peptidyl fragmentswhich can function as either agonists or antagonists of a wild-type(e.g., “naturally occurring”) BCL-xγ protein.

In another embodiment, an isolated or recombinant BCL-xγ proteinincludes a sequence corresponding to a γ domain (i.e., amino acids185–235 of SEQ ID NO:2) and having at least about 60% homology to thatsequence and has anti-apoptotic activity. In yet another embodiment, anisolated or recombinant BCL-xγ protein includes a sequence at leastabout 70% homologous to a γ domain of a BCL-xγ protein and hasanti-apoptotic activity. In a preferred embodiment, an isolated orrecombinant BCL-xγ protein includes a sequence at least about 80%homologous to a γ domain of a BCL-xγ protein and has anti-apoptoticactivity. In another preferred embodiment, an isolated or recombinantBCL-xγ protein includes a sequence at least about 90% homologous to a γdomain of a BCL-xγ protein and has anti-apoptotic activity. In aparticularly preferred embodiment an isolated or recombinant BCL-xγprotein includes a γ domain of a BCL-xγ protein such as that shown inSEQ ID NO:2 amino acids 185–235 and has anti-apoptotic activity.

In another embodiment, a BCL-xγ nucleic acid molecule encodes anintracellular protein containing an ankyrin-like domain which isanti-apoptotic. In one embodiment, a BCL-xγ nucleic acid moleculeencodes an intracellular protein with a consensus ankyrin domain shownin the sequence NXXXXXXGXTPLXX (SEQ ID NO: 25) which is anti-apoptotic.In a preferred embodiment, a BCL-xγ nucleic acid molecule encodes aprotein with the ankyrin domain shown in amino acids 185–217 of SEQ IDNO:2, and which is intracellular and anti-apoptotic.

In certain preferred embodiments, the invention features a purified orrecombinant BCL-xγ protein having a calculated molecular weight ofapproximately 26,122 kD. It will be understood that certainpost-translational modifications can increase the apparent molecularweight of the BCL-xγ protein relative to the unmodified polypeptidechain. The BCL-xγ protein migrates with an apparent molecular weight of33,000 kD.

This invention further provides a method for generating sets ofcombinatorial mutants of the subject BCL-xγ proteins as well astruncation mutants, and is especially useful for identifying potentialvariant sequences (e.g. homologs) that modulate a BCL-xγ bioactivity.The purpose of screening such combinatorial libraries is to generate,for example, novel BCL-xγ homologs which can act as either agonists orantagonist, or alternatively, possess novel activities all together. Toillustrate, combinatorially-derived homologs can be generated to have anincreased potency relative to a naturally occurring form of the protein.

Likewise, BCL-xγ homologs can be generated by the present combinatorialapproach to selectively inhibit (antagonize) naturally occurring BCL-xγ.Moreover, manipulation of certain domains of BCL-xγ by the presentmethod can provide domains more suitable for use in fusion proteins.

In one embodiment, a variegated library of BCL-xγ variants is generatedby combinatorial mutagenesis at the nucleic acid level, and is encodedby a variegated gene library. For instance, a mixture of syntheticoligonucleotides can be enzymatically ligated into gene sequences suchthat the degenerate set of potential BCL-xγ sequences are expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins (e.g. for phage display) containing the set of BCL-xγ sequencestherein.

There are many ways by which such libraries of potential BCL-xγ homologscan be generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then ligated into anappropriate expression vector. The purpose of a degenerate set of genesis to provide, in one mixture, all of the sequences encoding the desiredset of potential BCL-xγ sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rdCleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevier pp273–289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura etal. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al. (1990) Science 249:386–390;Roberts et al. (1992) PNAS 89:2429–2433; Devlin et al. (1990) Science249: 404–406; Cwirla et al. (1990) PNAS 87: 6378–6382; as well as U.S.Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

Likewise, a library of coding sequence fragments can be provided for aBCL-xγ clone in order to generate a variegated population of BCL-xγfragments for screening and subsequent selection of bioactive fragments.A variety of techniques are known in the art for generating suchlibraries, including chemical synthesis. In one embodiment, a library ofcoding sequence fragments can be generated by (i) treating a doublestranded PCR fragment of a bcl-xγ coding sequence with a nuclease underconditions wherein nicking occurs only about once per molecule; (ii)denaturing the double stranded DNA; (iii) renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products; (iv) removing single stranded portions fromreformed duplexes by treatment with S1 nuclease; and (v) ligating theresulting fragment library into an expression vector. By this exemplarymethod, an expression library can be derived which codes for N-terminal,C-terminal and internal fragments of various sizes.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of BCL-xγ homologs. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Each of the illustrative assays described beloware amenable to high through-put analysis as necessary to screen largenumbers of degenerate BCL-xγ sequences created by combinatorialmutagenesis techniques.

In one embodiment, cell based assays can be exploited to analyze thevariegated BCL-xγ library. For instance, the library of expressionvectors can be transfected into a T cell line, preferably a T cell linethat does not express BCL-xγ. The transfected cells are then treated toinduce apoptosis, e.g., with anti-CD3, and the effect of the BCL-xγmutant can be detected, e.g. cell viability. Plasmid DNA can then berecovered from the cells which score for inhibition, or alternatively,potentiation of a BCL-xγ activity, and the individual clones furthercharacterized.

Combinatorial mutagenesis has a potential to generate very largelibraries of mutant proteins, e.g., in the order of 10²⁶ molecules.Combinatorial libraries of this size can be screened using a variety oftechniques, e.g., recrusive ensemble mutagenesis (REM) (Arkin andYourvan, 1992, PNAS USA 89:7811–7815; Yourvan et al., 1992, ParallelProblem Solving from Nature, 2., In Maenner and Manderick, eds., ElsevirPublishing Co., Amsterdam, pp. 401–410; Delgrave et al., 1993, ProteinEngineering 6(3):327–331).

The invention also provides for reduction of the mammalian BCL-xγproteins to generate mimetics, e.g. peptide or non-peptide agents, whichare able to disrupt binding of a mammalian BCL-xγ protein of the presentinvention with binding proteins or interactors. Thus, such mutagenictechniques as described above are also useful to map the determinants ofthe BCL-xγ proteins which participate in protein-protein interactions.Such interactions can be involved in, for example, binding of thesubject mammalian BCL-xγ protein to proteins which may function upstream(including both activators and repressors of its activity) or downstreamof the BCL-xγ protein, whether they are positively or negativelyregulated by it. To illustrate, the critical residues of a subjectBCL-xγ protein which are involved in molecular recognition of interactorproteins upstream or downstream of a BCL-xγ (such as, for example BH1domains, BH2 domains, or ankyrin binding domains) can be determined andused to generate BCL-xγ-derived peptidomimetics which competitivelyinhibit binding of the naturally occurring BCL-xγ protein to thatmoiety. By employing, for example, scanning mutagenesis to map the aminoacid residues of each of the subject BCL-xγ proteins which are involvedin binding other extracellular proteins, peptidomimetic modulatingagents can be generated which mimic those residues of the BCL-xγ proteinwhich facilitate the interaction. Such mimetics may then be used tointerfere with the normal function of a BCL-xγ protein. For example,non-hydrolyzable peptide analogs of such residues can be generated usingbenzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry andBiology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry andBiology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988), substituted γ lactani rings (Garvey et al. in Peptides: Chemistryand Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988), keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem29:295; and Ewenson et al. in Peptides: Structure and Function(Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co.Rockland, Ill., 1985), b-turn dipeptide cores (Nagai et al. (1985)Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc Perkin Trans1:1231), and b-aminoalcohols (Gordon et al. (1985) Biochem Biophys ResCommun126:419; and Dann et al. (1986) Biochem Biophys Res Commun134:71).

In another embodiment, the coding sequences for the protein can beincorporated as a part of a fusion gene including a nucleotide sequenceencoding a different protein to generate a fusion protein or chimericprotein.

A “chimeric protein” or “fusion protein” is a fusion of a first aminoacid sequence encoding one of the subject mammalian BCL-xγ proteins witha second amino acid sequence defining a domain (e.g. protein portion)foreign to and not substantially homologous with any domain of one ofthe mammalian BCL-xγ proteins. A chimeric protein may present a foreigndomain which is found (albeit in a different protein) in an organismwhich also expresses the first protein, or it may be an “interspecies”,“intergenic”, etc. fusion of protein structures expressed by differentkinds of organisms. In general, a fusion protein can be represented bythe general formula X-BCL-xγ-Y, wherein BCL-xγ represents a portion ofthe protein which is derived from one of the mammalian BCL-xγ proteins,and X and Y are independently absent or represent amino acid sequenceswhich are not related to one of the mammalian BCL-xγ sequences in anorganism, including naturally occurring mutants.

Fusion proteins can also facilitate the expression of proteins, andaccordingly, can be used in the expression of the mammalian BCL-xγproteins of the present invention. For example, BCL-xγ proteins can begenerated as glutathione-S-transferase (GST-fusion) proteins. SuchGST-fusion proteins can enable easy purification of the BCL-xγ protein,as for example by the use of glutathione-derivatized matrices (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.(N.Y.: John Wiley & Sons, 1991)).

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant protein, canallow purification of the expressed fusion protein by affinitychromatography using a Ni2+ metal resin. The purification leadersequence can then be subsequently removed by treatment with enterokinaseto provide the purified protein (e.g., see Hochuli et al. (1987) J.Chromatography 411:177; and Janknecht et al. PNAS 88:8972).

Techniques for making fusion genes are known to those skilled in theart. Essentially, the joining of various DNA fragments coding fordifferent protein sequences is performed in accordance with conventionaltechniques, employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed to generate a chimeric genesequence (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al. John Wiley & Sons: 1992).

In preferred embodiments, fusion proteins of the present inventioncontain a detectable label or a matrix binding domain.

The preparation of fusion proteins is often desirable when producing animmunogenic fragment of a BCL-xγ protein. For example, the VP6 capsidprotein of rotavirus can be used as an immunologic carrier protein forportions of the BCL-xγ protein, either in the monomeric form or in theform of a viral particle. The nucleic acid sequences corresponding tothe portion of a subject BCL-xγ protein to which antibodies are to beraised can be incorporated into a fusion gene construct which includescoding sequences for a late vaccinia virus structural protein to producea set of recombinant viruses expressing fusion proteins comprisingBCL-xγ epitopes as part of the virion. It has been demonstrated with theuse of immunogenic fusion proteins utilizing the Hepatitis B surfaceantigen fusion proteins that recombinant Hepatitis B virions can beutilized in this role as well. Similarly, chimeric constructs coding forfusion proteins containing a portion of a BCL-xγ protein and thepoliovirus capsid protein can be created to enhance immunogenicity ofthe set of polypeptide antigens (see, for example, EP Publication NO:0259149; and Evans et al. (1989) Nature 339:385; Huang et al. (1988) J.Virol. 62:3855; and Schlienger et al. (1992) J. Virol. 66:2).

The Multiple Antigen Peptide system for peptide-based immunization canalso be utilized to generate an immunogen, wherein a desired portion ofa BCL-xγ protein is obtained directly from organo-chemical synthesis ofthe peptide onto an oligomeric branching lysine core (see, for example,Posnett et al. (1988) JBC 263:1719 and Nardelli et al. (1992) J.Immunol. 148:914). Antigenic determinants of BCL-xγ proteins can also beexpressed and presented by bacterial cells.

IV. Antibodies.

Another aspect of the invention pertains to an antibody specificallyreactive with a mammalian BCL-xγ protein. For example, by usingimmunogens derived from a BCL-xγ protein, e.g. based on the cDNAsequences, anti-protein/anti-peptide antisera or monoclonal antibodiescan be made by standard protocols (See, for example, Antibodies: ALaboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press:1988)). A mammal, such as a mouse, a hamster or rabbit can be immunizedwith an immunogenic form of the peptide (e.g., a mammalian BCL-xγprotein or an antigenic fragment which is capable of eliciting anantibody response, or a fusion protein as described above). Techniquesfor conferring immunogenicity on a protein or peptide includeconjugation to carriers or other techniques well known in the art. Animmunogenic portion of a BCL-xγ protein can be administered in thepresence of adjuvant. The progress of immunization can be monitored bydetection of antibody titers in plasma or serum. Standard ELISA or otherimmunoassays can be used with the immunogen as antigen to assess thelevels of antibodies. In a preferred embodiment, the subject antibodiesare immunospecific for antigenic determinants of a BCL-xγ protein of amammal, e.g. antigenic determinants of a protein represented by SEQ IDNO:2.

Following immunization of an animal with an antigenic preparation of aBCL-xγ polypeptide, anti-BCL-xγ antisera can be obtained and, ifdesired, polyclonal anti-BCL-xγ antibodies isolated from the serum. Toproduce monoclonal antibodies, antibody-producing cells (lymphocytes)can be harvested from an immunized animal and fused by standard somaticcell fusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, aninclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495–497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77–96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a mammalian BCL-xγprotein of the present invention and monoclonal antibodies isolated froma culture comprising such hybridoma cells.

As an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal anti-BCL-xγ antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with BCL-xγ to thereby isolateimmunoglobulin library members that bind BCL-xγ. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al.International Publication No. WO 92/18619; Dower et al. InternationalPublication No. WO 91/17271; Winter et al. International Publication WO92/20791; Markland et al. International Publication No. WO 92/15679;Breitling et al. International Publication WO 93/01288; McCafferty etal. International Publication No. WO 92/01047; Garrard et al.International Publication No. WO 92/09690; Ladner et al. InternationalPublication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology9:1370–1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81–85; Huse etal. (1989) Science 246:1275–1281; Griffiths et al. (1993) EMBO J12:725–734; Hawkins et al. (1992) J Mol Biol 226:889–896; Clarkson etal. (1991) Nature 352:624–628; Gram et al. (1992) PNAS 89:3576–3580;Garrad et al. (1991) Bio/Technology 9:1373–1377; Hoogenboom et al.(1991) Nuc Acid Res 19:4133–4137; Barbas et al. (1991) PNAS88:7978–7982; and McCafferty et al. Nature (1990) 348:552–554.

Additionally, recombinant anti-BCL-xγ antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Patent Publication PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT Application WO 86/01533; Cabilly et al. U.S. Pat. No.4,816,567; Cabilly et al. European Patent Application 125,023; Better etal. (1988) Science 240:1041–1043; Liu et al. (1987) PNAS 84:3439–3443;Liu et al. (1987) J. Immunol. 139:3521–3526; Sun et al. (1987) PNAS84:214–218; Nishimura et al. (1987) Canc. Res. 47:999–1005; Wood et al.(1985) Nature 314:446–449; and Shaw et al. (1988) J. Natl Cancer Inst.80:1553–1559); Morrison, S. L. (1985) Science 229:1202–1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552–525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol 141:4053–4060.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the subjectmammalian BCL-xγ proteins. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. For example, F(ab)₂fragments can be generated by treating antibody with pepsin. Theresulting F(ab)₂ fragment can be treated to reduce disulfide bridges toproduce Fab fragments. The antibody of the present invention is furtherintended to include bispecific and chimeric molecules having affinityfor a BCL-xγ protein conferred by at least one CDR region of theantibody.

Antibodies which specifically bind BCL-xγ epitopes can also be used inimmunohistochemical staining of tissue samples in order to evaluate theabundance and pattern of expression of each of the subject BCL-xγproteins. Anti-BCL-xγ antibodies can be used diagnostically inimmuno-precipitation and immuno-blotting to detect and evaluate BCL-xγprotein levels in tissue as part of a clinical testing procedure.Likewise, the ability to monitor BCL-xγ protein levels in an individualcan allow determination of the efficacy of a given treatment regimen foran individual afflicted with such a disorder. Diagnostic assays usinganti-BCL-xγ antibodies can include, for example, immunoassays designedto aid in early diagnosis of a degenerative disorder, particularly oneswhich are manifest at birth. Diagnostic assays using anti-BCL-xγ proteinantibodies can also include immunoassays designed to aid in earlydiagnosis and phenotyping neoplastic or hyperplastic disorders.

Another application of anti-BCL-xγ antibodies of the present inventionis in the immunological screening of cDNA libraries constructed inexpression vectors such as λgt11, λgt18–23, λZAP, and λORF8. Messengerlibraries of this type, having coding sequences inserted in the correctreading frame and orientation, can produce fusion proteins. Forinstance, λgt11 will produce fusion proteins whose amino termini consistof β-galactosidase amino acid sequences and whose carboxy terminiconsist of a foreign polypeptide. Antigenic epitopes of a BCL-xγprotein, e.g. other orthologs of a particular BCL-xγ protein or otherparalogs from the same species, can then be detected with antibodies,as, for example, reacting nitrocellulose filters lifted from infectedplates with anti-BCL-xγ antibodies. Positive phage detected by thisassay can then be isolated from the infected plate. Thus, the presenceof BCL-xγ homologs can be detected and cloned from other animals, as canalternate isoforms (including splicing variants) from humans.

In certain embodiments, it will be desirable to attach a label group tothe subject antibodies to facilitate detection. One means for labelingan anti-BCL-xγ protein specific antibody is via linkage to an enzyme anduse in an enzyme immunoassay (EIA) (Voller, “The Enzyme LinkedImmunosorbent Assay (ELISA)”, Diagnostic Horizons 2:1–7, 1978,Microbiological Associates Quarterly Publication, Walkersville, Md.;Voller, et al., J. Clin. Pathol. 31:507–520 (1978); Butler, Meth.Enzymol. 73:482–523 (1981); Maggio, (ed.) Enzyme Immunoassay, CRC Press,Boca Raton, Fla., 1980; Ishikawa, et al., (eds.) Enzyme Immunoassay,Kgaku Shoin, Tokyo, 1981). The enzyme which is bound to the antibodywill react with an appropriate substrate, preferably a chromogenicsubstrate, in such a manner as to produce a chemical moiety which can bedetected, for example, by spectrophotometric, fluorimetric or by visualmeans. Enzymes which can be used to detectably label the antibodyinclude, but are not limited to, malate dehydrogenase, staphylococcalnuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect fingerprint gene wild typeor mutant peptides through the use of a radioimmunoassay (RIA) (see, forexample, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March, 1986, which is incorporated by reference herein). The radioactiveisotope can be detected by such means as the use of a γ counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected. Among the mostcommonly used fluorescent labeling compounds are fluoresceinisothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as 152Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting luminescence that arises duringthe course of a chemical reaction. Examples of particularly usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

V. Methods of the Invention

Drug Screening Assays

The present invention provides for assays which can be used to screenfor modulating agents, including BCL-xγ homologs, which are eitheragonists or antagonists of the normal cellular function of the subjectBCL-xγ polypeptides. For example, the invention provides a method inwhich an indicator composition is provided which has a BCL-xγ proteinhaving BCL-xγ anti-apoptotic activity. The indicator composition can becontacted with a test compound. The effect of the test compound onBCL-xγ anti-apoptotic activity, as measured by a change in the indicatorcomposition, can then be determined to thereby identify a compound thatmodulates the anti-apoptotic activity of a BCL-xγ protein. Astatistically significant change, such as a decrease or increase, in thelevel of BCL-xγ anti-apoptotic activity in the presence of the testcompound (relative to what is detected in the absence of the testcompound) is indicative of the test compound being a BCL-xγ modulatingagent. The indicator composition can be, for example, a cell or a cellextract. In one embodiment, BCL-xγ anti-apoptotic activity is assessedas described in Example 9.

In many drug screening programs which test libraries of modulatingagents and natural extracts, high throughput assays are desirable inorder to maximize the number of modulating agents surveyed in a givenperiod of time. Assays which are performed in cell-free systems, such asmay be derived with purified or semi-purified proteins, are oftenpreferred as “primary” screens in that they can be generated to permitrapid development and relatively easy detection of an alteration in amolecular target which is mediated by a test modulating agent. Moreover,the effects of cellular toxicity and/or bioavailability of the testmodulating agent can be generally ignored in the in vitro system, theassay instead being focused primarily on the effect of the drug on themolecular target as may be manifest in an alteration of binding affinitywith upstream or downstream elements.

In an exemplary screening assay of the present invention, the modulatingagent of interest is cotacted with interactor proteins which mayfunction upstream (including both activators and repressors of itsactivity) or to proteins which may function downstream of the BCL-xγprotein, whether they are positively or negatively regulated by it. Tothe mixture of the modulating agent and the upstream or downstreamelement is then added a composition containing a BCL-xγ protein.Detection and quantification of the interaction of BCL-xγ with it'supstream or downstream elements provide a means for determining amodulating agent's efficacy at inhibiting (or potentiating) complexformation between BCL-xγ and the BCL-xγ-binding elements. The term“interact” as used herein is meant to include detectable interactionsbetween molecules, such as can be detected using, for example, a yeasttwo hybrid assay. The term interact is also meant to include “binding”interactions between molecules. Interactions may be protein-protein orprotein-nucleic acid in nature.

The efficacy of the modulating agent can be assessed by generating doseresponse curves from data obtained using various concentrations of thetest modulating agent. Moreover, a control assay can also be performedto provide a baseline for comparison. In the control assay, isolated andpurified BCL-xγ protein is added to a composition containing theBCL-xγ-binding element, and the formation of a complex is quantitated inthe absence of the test modulating agent.

Complex formation between the BCL-xγ protein and a BCL-xγ bindingelement may be detected by a variety of techniques. Modulation of theformation of complexes can be quantitated using, for example, detectablylabeled proteins such as radiolabeled, fluorescently labeled, orenzymatically labeled BCL-xγ proteins, by immunoassay, or bychromatographic detection.

Typically, it will be desirable to immobilize either BCL-xγ or itsbinding protein to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Binding of BCL-xγ to an upstream or downstreamelement, in the presence and absence of a candidate agent, can beaccomplished in any vessel suitable for containing the reactants.Examples include microtitre plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows the protein to be bound to a matrix. For example,glutathione-S-transferase/BCL-xγ (GST/BCL-xγ) fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates, e.g. an ³⁵S-labeled, and the testmodulating agent, and the mixture incubated under conditions conduciveto complex formation, e.g., at physiological conditions for salt and pH,though slightly more stringent conditions may be desired. Followingincubation, the beads are washed to remove any unbound label, and thematrix immobilized and radiolabel determined directly (e.g. beads placedin scintilant), or in the supernatant after the complexes aresubsequently dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofBCL-xγ-binding protein found in the bead fraction quantitated from thegel using standard electrophoretic techniques such as described in theappended examples.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, either BCL-xγ orits cognate binding protein can be immobilized utilizing conjugation ofbiotin and streptavidin. For instance, biotinylated BCL-xγ molecules canbe prepared from biotin-NHS (N-hydroxy-succinimide) using techniqueswell known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical). Alternatively, antibodies reactive withBCL-xγ but which do not interfere with binding of upstream or downstreamelements can be derivatized to the wells of the plate, and BCL-xγtrapped in the wells by antibody conjugation. As above, preparations ofa BCL-xγ-binding protein and a test modulating agent are incubated inthe BCL-xγ-presenting wells of the plate, and the amount of complextrapped in the well can be quantitated. Exemplary methods for detectingsuch complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the BCL-xγ binding element, or which arereactive with BCL-xγ protein and compete with the binding element; aswell as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the binding element, either intrinsic orextrinsic activity. In the instance of the latter, the enzyme can bechemically conjugated or provided as a fusion protein with theBCL-xγ-BP. To illustrate, the BCL-xγ-BP can be chemically cross-linkedor genetically fused with horseradish peroxidase, and the amount ofprotein trapped in the complex can be assessed with a chromogenicsubstrate of the enzyme, e.g. 3,3′-diamino-benzadine terahydrochlorideor 4-chloro-1-napthol. Likewise, a fusion protein comprising the proteinand glutathione-S-transferase can be provided, and complex formationquantitated by detecting the GST activity using1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).

For processes which rely on immunodetection for quantitating one of theproteins trapped in the complex, antibodies against the protein, such asanti-BCL-xγ antibodies, can be used. Alternatively, the protein to bedetected in the complex can be “epitope tagged” in the form of a fusionprotein which includes, in addition to the BCL-xγ sequence, a secondprotein for which antibodies are readily available (e.g. from commercialsources). For instance, the GST fusion proteins described above can alsobe used for quantification of binding using antibodies against the GSTmoiety. Other useful epitope tags include myc-epitopes (e.g., seeEllison et al. (1991) J Biol Chem 266:21150–21157) which includes a10-residue sequence from c-myc, as well as the pFLAG system(International Biotechnologies, Inc.) or the pEZZ-protein A system(Pharamacia, N.J).

In addition to cell-free assays, such as described above, the readilyavailable source of mammalian BCL-xγ proteins provided by the presentinvention also facilitates the generation of cell-based assays foridentifying small molecule agonists/antagonists and the like. Forexample, cells can be caused to overexpress a recombinant BCL-xγ proteinin the presence and absence of a test modulating agent of interest, withthe assay scoring for modulation in BCL-xγ responses by the target- cellmediated by the test agent. As with the cell-free assays, modulatingagents which produce a statistically significant change inBCL-xγ-dependent responses (either inhibition or potentiation) can beidentified. In an illustrative embodiment, the expression or activity ofa BCL-xγ is modulated in cells and the effects of modulating agents ofinterest on the readout of interest (such as apoptosis) are measured.For example, the expression of genes which are up- or down-regulated inresponse to a T cell receptor-mediated signal cascade can be assayed. Inpreferred embodiments, the regulatory regions of such genes, e.g., the5′ flanking promoter and enhancer regions, are operatively linked to amarker (such as luciferase) which encodes a gene product that can bereadily detected.

Monitoring the influence of modulating agents on cells may be appliednot only in basic drug screening, but also in clinical trials. In suchclinical trials, the expression of a panel of genes may be used as a“read out” of a particular drug's therapeutic effect.

In another aspect of the invention, the subject BCL-xγ proteins can beused to generate a “two hybrid” assay (see, for example, U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223–232; Madura et al. (1993) JBiol Chem 268:12046–12054; Bartel et al. (1993) Biotechniques14:920–924; Iwabuchi et al. (1993) Oncogene 8:1693–1696; and BrentWO94/10300), for isolating coding sequences for other cellular proteinswhich bind to or interact with BCL-xγ (“BCL-xγ-binding proteins” or“BCL-xγ-bp”). Such BCL-xγ-binding proteins would likely be regulators ofBCL-xγ bioactivity.

Briefly, the two hybrid assay relies on reconstituting in vivo afunctional transcriptional activator protein from two separate fusionproteins. In particular, the method makes use of chimeric genes whichexpress hybrid proteins. To illustrate, a first hybrid gene comprisesthe coding sequence for a DNA-binding domain of a transcriptionalactivator fused in frame to the coding sequence for a BCL-xγ protein.The second hybrid protein encodes a transcriptional activation domainfused in frame to a sample gene from a cDNA library. If the bait andsample hybrid proteins are able to interact, e.g., form aBCL-xγ-dependent complex, they bring into close proximity the DNAbinding domain and the activation domain of the transcriptionalactivator. This proximity is sufficient to cause transcription of areporter gene which is operatively linked to a transcriptionalregulatory site responsive to the transcriptional activator, andexpression of the reporter gene can be detected and used to score forthe interaction of the BCL-xγ and sample proteins.

Diagnostic and Prognostic Assays

The present method provides a method for determining if a subject is atrisk for a disorder characterized by aberrant cell proliferation orapoptosis. In preferred embodiments, the methods can be characterized ascomprising detecting, in a sample of cells from the subject, thepresence or absence of a genetic lesion characterized by at least one of(i) an alteration affecting the integrity of a gene encoding aBCL-xγ-protein, or (ii) the misregulation or (iii) aberrantpost-translational modification of the bcl-xγ gene. To illustrate, suchgenetic lesions can be detected by ascertaining the existence of atleast one of (i) a deletion of one or more nucleotides from a bcl-xγgene, (ii) an addition of one or more nucleotides to a bcl-xγ gene,(iii) a substitution of one or more nucleotides of a bcl-xγ gene, (iv) agross chromosomal rearrangement of a BCL-xγ gene, (v) a gross alterationin the level of a messenger RNA transcript of a bcl-xγ gene, (vii)aberrant modification of a BCL-xγ gene, such as of the methylationpattern of the genomic DNA, (vii) the presence of a non-wild typesplicing pattern of a messenger RNA transcript of a bcl-xγ gene, (viii)a non-wild type level of a BCL-xγ-protein, (ix) allelic loss of a bcl-xγgene, and (x) inappropriate post-translational modification of aBCL-xγ-protein. As set out below, the present invention provides a largenumber of assay techniques for detecting lesions in a bcl-xγ gene, andimportantly, provides the ability to discern between different molecularcauses underlying BCL-xγ-dependent aberrant cell growth, proliferationand/or differentiation.

As discussed in more detail below, the probes of the present inventioncan also be used as a part of a diagnostic test kit for identifyingcells or tissue which misexpress a BCL-xγ protein, such as by measuringa level of a BCL-xγ-encoding nucleic acid in a sample of cells from apatient; e.g. detecting BCL-xγ mRNA levels or determining whether agenomic bcl-xγ gene has been mutated or deleted. Briefly, nucleotideprobes can be generated from the subject bcl-xγ genes which facilitatehistological screening of intact tissue and tissue samples for thepresence (or absence) of BCL-xγ-encoding transcripts. Similar to thediagnostic uses of anti-BCL-xγ antibodies (described in detail below),the use of probes directed to BCL-xγ messages, or to genomic bcl-xγsequences, can be used for both predictive and therapeutic evaluation ofallelic mutations which might be manifest in certain disorders. Used inconjunction with immunoassays as described herein, the oligonucleotideprobes can help facilitate the determination of the molecular basis fora disorder which may involve some abnormality associated with expression(or lack thereof) of a BCL-xγ protein. For instance, variation inpolypeptide synthesis can be differentiated from a mutation in a codingsequence.

In an exemplary embodiment, a nucleic acid composition is provided whichcontains an oligonucleotide probe previously described. The nucleic acidof a cell is rendered accessible for hybridization, the probe is exposedto nucleic acid of the sample, and the hybridization of the probe to thesample nucleic acid is detected. Such techniques can be used to detectlesions at either the genomic or mRNA level, including deletions,substitutions, etc., as well as to determine mRNA transcript levels.

In certain embodiments, detection of the lesion comprises utilizing theprobe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077–1080; and Nakazawa et al. (1994) PNAS91:360–364), the latter of which can be particularly useful fordetecting point mutations in the bcl-xγ-gene (see Abravaya et al. (1995)Nuc Acid Res 23:675–682). In an illustrative embodiment, the methodincludes the steps of (i) collecting a sample of cells from a patient,(ii) isolating nucleic acid (e.g., genomic, mRNA or both) from the cellsof the sample, (iii) contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a bcl-xγ gene under conditionssuch that hybridization and amplification of the bcl-xγ-gene (ifpresent) occurs, and (iv) detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA87:1874–1878), transcriptional amplification system (Kwoh, D. Y. et al.,1989, Proc. Natl. Acad. Sci. USA 86:1173–1177), Q-Beta Replicase(Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In another embodiment of the subject assay, mutations in a bcl-xγ genefrom a sample cell are identified by alterations in restriction enzymecleavage patterns. For example, sample and control DNA is isolated,amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis. Moreover, the use of sequence specific ribozymes (see,for example, U.S. Pat. No. 5,498,531) can be used to score for thepresence of specific mutations by development or loss of a ribozymecleavage site.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the bcl-xγ gene anddetect mutations by comparing the sequence of the sample BCL-xγ with thecorresponding wild-type (control) sequence. Exemplary sequencingreactions include those based on techniques developed by Maxim andGilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger (Sanger et al(1977) Proc. Nat. Acad. Sci 74:5463). Any of a variety of automatedsequencing procedures may be utilized when performing the subject assays(Biotechniques (1995) 19:448), including by sequencing by massspectrometry (see, for example PCT publication WO 94/16101; Cohen et al.(1996) Adv Chromatogr 36:127–162; and Griffin et al. (1993) Appl BiochemBiotechnol 38:147–159). It will be evident to one skilled in the artthat, for certain embodiments, the occurrence of only one, two or threeof the nucleic acid bases need be determined in the sequencing reaction.For instance, A-tract sequencing where only one nucleic acid isdetected, can be carried out.

In a further embodiment, protection from cleavage agents (such as anuclease, hydroxylamine or osmium tetroxide and with piperidine) can beused to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes(Myers, et al. (1985) Science 230:1242). In general, the art techniqueof “mismatch cleavage” starts by providing heteroduplexes formed byhybridizing (labelled) RNA or DNA containing the wild-type BCL-xγsequence with potentially mutant RNA or DNA obtained from a tissuesample. The double-stranded duplexes are treated with an agent whichcleaves single-stranded regions of the duplex such as which will existdue to basepair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digesting the mismatchedregions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can betreated with hydroxylamine or osmium tetroxide and with piperidine inorder to digest mismatched regions. After digestion of the mismatchedregions, the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, for example,Cotton et al (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al(1992) Methods Enzymol. 217:286–295. In a preferred embodiment, thecontrol DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in BCL-xγ cDNAs obtained fromsamples of cells. For example, the mutΥ enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657–1662).According to an exemplary embodiment, a probe based on a BCL-xγsequence, e.g., a wild-type BCL-xγ sequence, is hybridized to a cDNA orother DNA product from a test cell(s). The duplex is treated with a DNAmismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. See, for example,U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in bcl-xγ genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766, see also Cotton(1993) Mutat Res 285:125–144; and Hayashi (1992) Genet Anal Tech Appl9:73–79). Single-stranded DNA fragments of sample and control BCL-xγnucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may belabelled or detected with labelled probes. The sensitivity of the assaymay be enhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al (1985)Nature 313:495). When DGGE is used as the method of analysis, DNA willbe modified to insure that it does not completely denature, for exampleby adding a GC clamp of approximately 40 bp of high-melting GC-rich DNAby PCR. In a further embodiment, a temperature gradient is used in placeof a denaturing agent gradient to identify differences in the mobilityof control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al (1989) Proc. Natl Acad. Sci USA86:6230). Such allele specific oligonucleotide hybridization techniquesmay be used to test one mutation per reaction when oligonucleotides arehybridized to PCR amplified target DNA or a number of differentmutations when the oligonucleotides are attached to the hybridizingmembrane and hybridized with labelled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al(1989) Nucleic Acids Res. 17:2437–2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238. Inaddition, it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a BCL-xγ gene.

Diagnostic procedures may also be performed in situ directly upon tissuesections (fixed and/or frozen) of patient tissue obtained from biopsiesor resections, such that no nucleic acid purification is necessary.Nucleic acid reagents may be used as probes and/or primers for such insitu procedures (see, for example, Nuovo, G. J., 1992, PCR in situhybridization: protocols and applications, Raven Press, NY).

In addition to methods which focus primarily on the detection of onenucleic acid sequence, profiles may also be assessed in such detectionschemes. Fingerprint profiles may be generated, for example, byutilizing a differential display procedure, Northern analysis and/orRT-PCR.

Antibodies directed against wild type or mutant BCL-xγ proteins, whichare discussed, above, may also be used in disease diagnostics andprognostics. Such diagnostic methods, may be used to detectabnormalities in the level of BCL-xγ protein expression, orabnormalities in the structure and/or tissue, cellular, or subcellularlocation of BCL-xγ protein. Structural differences may include, forexample, differences in the size, electronegativity, or antigenicity ofthe mutant BCL-xγ protein relative to the normal BCL-xγ protein. Proteinfrom the tissue or cell type to be analyzed may easily be detected orisolated using techniques which are well known to one of skill in theart, including but not limited to western blot analysis. For a detailedexplanation of methods for carrying out western blot analysis, seeSambrook et al, 1989, supra, at Chapter 18. The protein detection andisolation methods employed herein may also be such as those described inHarlow and Lane, for example, (Harlow, E. and Lane, D., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.), which is incorporated herein by reference inits entirety.

This can be accomplished, for example, by immunofluorescence techniquesemploying a fluorescently labeled antibody (see below) coupled withlight microscopic, flow cytometric, or fluorimetric detection. Theantibodies (or fragments thereof) useful in the present invention may,additionally, be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of BCL-xγ proteins. Insitu detection may be accomplished by removing a histological specimenfrom a patient, and applying thereto a labeled antibody of the presentinvention. The antibody (or fragment) is preferably applied byoverlaying the labeled antibody (or fragment) onto a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of the BCL-xγ protein, but also its distribution inthe examined tissue. Using the present invention, one of ordinary skillwill readily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

Often a solid phase support or carrier is used as a support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

Moreover, any of the above methods for detecting alterations in a bcl-xγgene or gene product can be used to monitor the course of treatment ortherapy.

Modulation of BCL-xγ Activity

Yet another aspect of the invention pertains to methods of modulatingBCL-xγ activity in a cell. The modulatory methods of the inventioninvolve contacting the cell with an agent that modulates BCL-xγ activitysuch that BCL-xγ activity in the cell is modulated. The agent may act bymodulating the activity of BCL-xγ protein in the cell or by modulatingtranscription of the BCL-xγ gene or translation of the BCL-xγ mRNA. Asused herein, the term “modulating” is intended to include inhibiting ordecreasing BCL-xγ activity and stimulating or increasing BCL-xγactivity. Accordingly, in one embodiment, the agent inhibits BCL-xγactivity. An inhibitory agent may function, for example, by directlyinhibiting BCL-xγ anti-apoptotic activity or by modulating a signalingpathway which negatively regulates BCL-xγ. In another embodiment, theagent stimulates BCL-xγ activity. A stimulatory agent may function, forexample, by directly stimulating BCL-xγ anti-apoptotic activity, or bymodulating a signaling pathway that leads to stimulation of BCL-xγactivity.

A. Inhibitory Agents

According to one modulatory method of the invention, BCL-xγ activity isinhibited in a cell by contacting the cell with an inhibitory agent.Inhibitory agents of the invention can be, for example, intracellularbinding molecules that act to inhibit the expression or activity ofBCL-xγ. As used herein, the term “intracellular binding molecule” isintended to include molecules that act intracellularly to inhibit theexpression or activity of a protein by binding to the protein itself, toa nucleic acid (e.g., an mRNA molecule) that encodes the protein or to asecond protein with which the first protein normally interacts (e.g., aBCL-xγ binding protein). Examples of intracellular binding molecules,described in further detail below, include antisense BCL-xγ nucleic acidmolecules (e.g., to inhibit translation of BCL-xγ mRNA), intracellularanti-BCL-xγ antibodies (e.g., to inhibit the activity of BCL-xγprotein), and dominant negative mutants of the BCL-xγ protein.

In one embodiment, an inhibitory agent of the invention is an antisensenucleic acid molecule that is complementary to a gene encoding BCL-xγ,or to a portion of said gene, or a recombinant expression vectorencoding said antisense nucleic acid molecule. The use of antisensenucleic acids to downregulate the expression of a particular protein ina cell is well known in the art (see e.g., Weintraub, H. et al.,Antisense RNA as a molecular tool for genetic analysis, Reviews—Trendsin Genetics, Vol. 1(1) 1986; Askari, F. K. and McDonnell, W. M. (1996)N. Eng. J. Med. 334:316–318; Bennett, M. R. and Schwartz, S. M. (1995)Circulation 92:1981–1993; Mercola, D. and Cohen, J. S. (1995) CancerGene Ther. 2:47–59; Rossi, J. J. (1995) Br. Med. Bull. 51:217–225;Wagner, R. W. (1994) Nature 372:333–335). An antisense nucleic acidmolecule comprises a nucleotide sequence that is complementary to thecoding strand of another nucleic acid molecule (e.g., an mRNA sequence)and accordingly is capable of hydrogen bonding to the coding strand ofthe other nucleic acid molecule. Antisense sequences complementary to asequence of an mRNA can be complementary to a sequence found in thecoding region of the mRNA, the 5′ or 3′ untranslated region of the mRNAor a region bridging the coding region and an untranslated region (e.g.,at the junction of the 5′ untranslated region and the coding region).Furthermore, an antisense nucleic acid can be complementary in sequenceto a regulatory region of the gene encoding the mRNA, for instance atranscription initiation sequence or regulatory element. Preferably, anantisense nucleic acid is designed so as to be complementary to a regionpreceding or spanning the initiation codon on the coding strand or inthe 3′ untranslated region of an mRNA. An antisense nucleic acid forinhibiting the expression of BCL-xγ protein in a cell can be designedbased upon the nucleotide sequence encoding the BCL-xγ protein (e.g.,SEQ ID NO: 1 or a portion thereof), constructed according to the rulesof Watson and Crick base pairing.

An antisense nucleic acid can exist in a variety of different forms. Forexample, the antisense nucleic acid can be an oligonucleotide that iscomplementary to only a portion of a BCL-xγ gene. Antisenseoligonucleotides can be constructed using chemical synthesis proceduresknown in the art. An antisense oligonucleotide can be chemicallysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids, e.g. phosphorothioatederivatives and acridine substituted nucleotides can be used. To inhibitBCL-xγ expression in cells in culture, one or more antisenseoligonucleotides can be added to cells in culture media, typically atabout 200 mg oligonucleotide/ml.

Alternatively, an antisense nucleic acid can be produced biologicallyusing an expression vector into which a nucleic acid has been subclonedin an antisense orientation (i.e., nucleic acid transcribed from theinserted nucleic acid will be of an antisense orientation to a targetnucleic acid of interest). Regulatory sequences operatively linked to anucleic acid cloned in the antisense orientation can be chosen whichdirect the expression of the antisense RNA molecule in a cell ofinterest, for instance promoters and/or enhancers or other regulatorysequences can be chosen which direct constitutive, tissue specific orinducible expression of antisense RNA. For example, for inducibleexpression of antisense RNA, an inducible eukaryotic regulatory system,such as the Tet system (e.g., as described in Gossen, M. and Bujard, H.(1992) Proc. Natl. Acad. Sci. USA 89:5547–5551; Gossen, M. et al. (1995)Science 268:1766–1769; PCT Publication No. WO 94/29442; and PCTPublication No. WO 96/01313) can be used. The antisense expressionvector is prepared as described above for recombinant expressionvectors, except that the cDNA (or portion thereof) is cloned into thevector in the antisense orientation. The antisense expression vector canbe in the form of, for example, a recombinant plasmid, phagemid orattenuated virus. The antisense expression vector is introduced intocells using a standard transfection technique, as described above forrecombinant expression vectors.

In another embodiment, an antisense nucleic acid for use as aninhibitory agent is a ribozyme. Ribozymes are catalytic RNA moleculeswith ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region (for reviews on ribozymes see e.g., Ohkawa, J. etal. (1995) J. Biochem. 118:251–258; Sigurdsson, S. T. and Eckstein, F.(1995) Trends Biotechnol. 13:286–289; Rossi, J. J. (1995) TrendsBiotechnol. 13:301–306; Kiehntopf, M. et al. (1995) J. Mol. Med.73:65–71). A ribozyme having specificity for BCL-xγ mRNA can be designedbased upon the nucleotide sequence of the BCL-xγ cDNA. For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thebase sequence of the active site is complementary to the base sequenceto be cleaved in a BCL-xγ mRNA. See for example U.S. Pat. Nos. 4,987,071and 5,116,742, both by Cech et al. Alternatively, BCL-xγ mRNA can beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See for example Bartel, D. and Szostak, J.W. (1993) Science 261: 1411–1418.

Another type of inhibitory agent that can be used to inhibit theexpression and/or activity of BCL-xγ in a cell is an intracellularantibody specific for the BCL-xγ protein. The use of intracellularantibodies to inhibit protein function in a cell is known in the art(see e.g., Carlson, J. R. (1988) Mol. Cell. Biol. 8:2638–2646; Biocca,S. et al. (1990) EMBO J. 9:101–108; Werge, T. M. et al. (1990) FEBSLetters 274:193–198; Carlson, J. R. (1993) Proc. Natl. Acad. Sci. USA90:7427–7428; Marasco, W. A. et al. (1993) Proc. Natl. Acad. Sci. USA90:7889–7893; Biocca, S. et al. (1994) Bio/Technology 12:396–399; Chen,S-Y. et al. (1994) Human Gene Therapy 5:595–601; Duan, L et al. (1994)Proc. Natl. Acad. Sci. USA 91:5075–5079; Chen, S-Y. et al. (1994) Proc.Natl. Acad. Sci. USA 91:5932–5936; Beerli, R. R. et al. (1994) J. Biol.Chem. 269:23931–23936; Beerli, R. R. et al. (1994) Biochem. Biophys.Res. Commun. 204:666–672; Mhashilkar, A. M. et al. (1995) EMBO J.14:1542–1551; Richardson, J. H. et al. (1995) Proc. Natl. Acad. Sci. USA92:3137–3141; PCT Publication No. WO 94/02610 by Marasco et al.; and PCTPublication No. WO 95/03832 by Duan et al.).

To inhibit protein activity using an intracellular antibody, arecombinant expression vector is prepared which encodes the antibodychains in a form such that, upon introduction of the vector into a cell,the antibody chains are expressed as a functional antibody in anintracellular compartment of the cell. For inhibition of BCL-xγ activityaccording to the inhibitory methods of the invention, an intracellularantibody that specifically binds the BCL-xγ protein is expressed in thecytoplasm of the cell. To prepare an intracellular antibody expressionvector, antibody light and heavy chain cDNAs encoding antibody chainsspecific for the target protein of interest, e.g., BCL-xγ, are isolated,typically from a hybridoma that secretes a monoclonal antibody specificfor the BCL-xγ protein. Hybridomas secreting anti-BCL-xγ monoclonalantibodies, or recombinant anti-BCL-xγ monoclonal antibodies, can beprepared as described above. Once a monoclonal antibody specific forBCL-xγ protein has been identified (e.g., either a hybridoma-derivedmonoclonal antibody or a recombinant antibody from a combinatoriallibrary), DNAs encoding the light and heavy chains of the monoclonalantibody are isolated by standard molecular biology techniques. Forhybridoma derived antibodies, light and heavy chain cDNAs can beobtained, for example, by PCR amplification or cDNA library screening.For recombinant antibodies, such as from a phage display library, cDNAencoding the light and heavy chains can be recovered from the displaypackage (e.g., phage) isolated during the library screening process.Nucleotide sequences of antibody light and heavy chain genes from whichPCR primers or cDNA library probes can be prepared are known in the art.For example, many such sequences are disclosed in Kabat, E. A., et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242 and in the “Vbase” human germline sequence database.

Once obtained, the antibody light and heavy chain sequences are clonedinto a recombinant expression vector using standard methods. To allowfor cytoplasmic expression of the light and heavy chains, the nucleotidesequences encoding the hydrophobic leaders of the light and heavy chainsare removed. An intracellular antibody expression vector can encode anintracellular antibody in one of several different forms. For example,in one embodiment, the vector encodes full-length antibody light andheavy chains such that a full-length antibody is expressedintracellularly. In another embodiment, the vector encodes a full-lengthlight chain but only the VH/CH1 region of the heavy chain such that aFab fragment is expressed intracellularly. In the most preferredembodiment, the vector encodes a single chain antibody (scFv) whereinthe variable regions of the light and heavy chains are linked by aflexible peptide linker (e.g., (Gly₄Ser)₃) and expressed as a singlechain molecule. To inhibit BCL-xγ activity in a cell, the expressionvector encoding the anti-BCL-xγ intracellular antibody is introducedinto the cell by standard transfection methods, as discussedhereinbefore.

Other inhibitory agents that can be used to inhibit the activity of aBCL-xγ protein are chemical compounds that inhibit BCL-xγ anti-apoptoticactivity. Such compounds can be identified using screening assays thatselect for such compounds, as described herein. Additionally oralternatively, compounds that inhibit BCL-xγ anti-apoptotic activity canbe designed using approaches known in the art.

B. Stimulatory Agents

According to another modulatory method of the invention, BCL-xγ activityis stimulated in a cell by contacting the cell with a stimulatory agent.Examples of such stimulatory agents include active BCL-xγ protein andnucleic acid molecules encoding BCL-xγ that are introduced into the cellto increase BCL-xγ activity in the cell. A preferred stimulatory agentis a nucleic acid molecule encoding a BCL-xγ protein, wherein thenucleic acid molecule is introduced into the cell in a form suitable forexpression of the active BCL-xγ protein in the cell. To express a BCL-xγprotein in a cell, typically a BCL-xγ cDNA is first introduced into arecombinant expression vector using standard molecular biologytechniques, as described herein. A BCL-xγ cDNA can be obtained, forexample, by amplification using the polymerase chain reaction (PCR) orby screening an appropriate cDNA library as described herein. Followingisolation or amplification of BCL-xγ cDNA, the DNA fragment isintroduced into an expression vector and transfected into target cellsby standard methods, as described herein.

Other stimulatory agents that can be used to stimulate the activity of aBCL-xγ protein are chemical compounds that stimulate BCL-xγ activity incells, such as compounds that enhance BCL-xγ anti-apoptotic activity.Such compounds can be identified using screening assays that select forsuch compounds, as described in detail above.

The modulatory methods of the invention can be performed in vitro (e.g.,by culturing the cell with the agent or by introducing the agent intocells in culture) or, alternatively, in vivo (e.g., by administering theagent to a subject or by introducing the agent into cells of a subject,such as by gene therapy). For practicing the modulatory method in vitro,cells can be obtained from a subject by standard methods and incubated(i.e., cultured) in vitro with a modulatory agent of the invention tomodulate BCL-xγ activity in the cells. For example, peripheral bloodmononuclear cells (PBMCs) can be obtained from a subject and isolated bydensity gradient centrifugation, e.g., with Ficoll/Hypaque. Specificcell populations can be depleted or enriched using standard methods. Forexample, monocytes/macrophages can be isolated by adherence on plastic.T cells can be enriched for example, by positive selection usingantibodies to T cell surface markers, for example by incubating cellswith a specific primary monoclonal antibody (mAb), followed by isolationof cells that bind the mAb using magnetic beads coated with a secondaryantibody that binds the primary mAb. Specific cell populations (e.g., Tcells) can also be isolated by fluorescence activated cell sortingaccording to standard methods. Monoclonal antibodies to T cell-specificsurface markers known in the art and many are commercially available. Ifdesired, cells treated in vitro with a modulatory agent of the inventioncan be readministered to the subject. For administration to a subject,it may be preferable to first remove residual agents in the culture fromthe cells before administering them to the subject. This can be done forexample by a Ficoll/Hypaque gradient centrifugation of the cells. Forfurther discussion of ex vivo genetic modification of cells followed byreadministration to a subject, see also U.S. Pat. No. 5,399,346 by W. F.Anderson et al.

For practicing the modulatory method in vivo in a subject, themodulatory agent can be administered to the subject such that BCL-xγactivity in cells of the subject is modulated. The term “subject” isintended to include living organisms in which an immune response can beelicited. Preferred subjects are mammals. Examples of subjects includehumans, monkeys, dogs, cats, mice, rats, cows, horses, goats and sheep.

For stimulatory or inhibitory agents that comprise nucleic acids(including recombinant expression vectors encoding BCL-xγ protein,antisense RNA, intracellular antibodies or dominant negativeinhibitors), the agents can be introduced into cells of the subjectusing methods known in the art for introducing nucleic acid (e.g., DNA)into cells in vivo. Examples of such methods encompass both non-viraland viral methods, including:

Direct Injection: Naked DNA can be introduced into cells in vivo bydirectly injecting the DNA into the cells (see e.g., Acsadi et al.(1991) Nature 332:815–818; Wolff et al. (1990) Science 247:1465–1468).For example, a delivery apparatus (e.g., a “gene gun”) for injecting DNAinto cells in vivo can be used. Such an apparatus is commerciallyavailable (e.g., from BioRad).

Cationic Lipids: Naked DNA can be introduced into cells in vivo bycomplexing the DNA with cationic lipids or encapsulating the DNA incationic liposomes. Examples of suitable cationic lipid formulationsinclude N-[-1-(2,3-dioleoyloxy)propyl]N,N,N-triethylammonium chloride(DOTMA) and a 1:1 molar ratio of1,2-dimyristyloxy-propyl-3-dimethylhydroxyethylammonium bromide (DMRIE)and dioleoyl phosphatidylethanolamine (DOPE) (see e.g., Logan, J. J. etal. (1995) Gene Therapy 2:38–49; San, H. et al. (1993) Human GeneTherapy 4:781–788).

Receptor-Mediated DNA Uptake: Naked DNA can also be introduced intocells in vivo by complexing the DNA to a cation, such as polylysine,which is coupled to a ligand for a cell-surface receptor (see forexample Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621; Wilson etal. (1992) J. Biol. Chem. 267:963–967; and U.S. Pat. No. 5,166,320).Binding of the DNA-ligand complex to the receptor facilitates uptake ofthe DNA by receptor-mediated endocytosis. A DNA-ligand complex linked toadenovirus capsids which naturally disrupt endosomes, thereby releasingmaterial into the cytoplasm can be used to avoid degradation of thecomplex by intracellular lysosomes (see for example Curiel et al. (1991)Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl.Acad. Sci. USA 90:2122–2126).

Retroviruses: Defective retroviruses are well characterized for use ingene transfer for gene therapy purposes (for a review see Miller, A. D.(1990) Blood 76:271). A recombinant retrovirus can be constructed havinga nucleotide sequences of interest incorporated into the retroviralgenome. Additionally, portions of the retroviral genome can be removedto render the retrovirus replication defective. The replicationdefective retrovirus is then packaged into virions which can be used toinfect a target cell through the use of a helper virus by standardtechniques. Protocols for producing recombinant retroviruses and forinfecting cells in vitro or in vivo with such viruses can be found inCurrent Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.)Greene Publishing Associates, (1989), Sections 9.10–9.14 and otherstandard laboratory manuals. Examples of suitable retroviruses includepLJ, pZIP, pWE and pEM which are well known to those skilled in the art.Examples of suitable packaging virus lines include ψcrip, ψCre, ψ2 andψAm. Retroviruses have been used to introduce a variety of genes intomany different cell types, including epithelial cells, endothelialcells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitroand/or in vivo (see for example Eglitis, et al. (1985) Science230:1395–1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA85:6460–6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA85:3014–3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA87:6141–6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA88:8039–8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377–8381; Chowdhury et al. (1991) Science 254:1802–1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640–7644; Kay etal. (1992) Human Gene Therapy 3:641–647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892–10895; Hwu et al. (1993) J. Immunol.150:4104–4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Retroviral vectors requiretarget cell division in order for the retroviral genome (and foreignnucleic acid inserted into it) to be integrated into the host genome tostably introduce nucleic acid into the cell. Thus, it may be necessaryto stimulate replication of the target cell.

Adenoviruses: The genome of an adenovirus can be manipulated such thatit encodes and expresses a gene product of interest but is inactivatedin terms of its ability to replicate in a normal lytic viral life cycle.See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld etal. (1991) Science 252:431–434; and Rosenfeld et al. (1992) Cell68:143–155. Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3,Ad7 etc.) are well known to those skilled in the art. Recombinantadenoviruses are advantageous in that they do not require dividing cellsto be effective gene delivery vehicles and can be used to infect a widevariety of cell types, including airway epithelium (Rosenfeld et al.(1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc.Natl. Acad. Sci. USA 89:6482–6486), hepatocytes (Herz and Gerard (1993)Proc. Natl. Acad. Sci. USA 90:2812–2816) and muscle cells (Quantin etal. (1992) Proc. Natl. Acad. Sci. USA 89:2581–2584). Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situations where introduced DNA becomes integrated intothe host genome (e.g., retroviral DNA). Moreover, the carrying capacityof the adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Berkner et al. cited supra;Haj-Ahmand and Graham (1986) J. Virol. 57:267). Mostreplication-defective adenoviral vectors currently in use are deletedfor all or parts of the viral E1 and E3 genes but retain as much as 80%of the adenoviral genetic material.

Adeno-Associated Viruses: Adeno-associated virus (AAV) is a naturallyoccurring defective virus that requires another virus, such as anadenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal. Curr. Topics in Micro. and Immunol. (1992) 158:97–129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349–356;Samulski et al. (1989) J. Virol. 63:3822–3828; and McLaughlin et al.(1989) J. Virol. 62:1963–1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985) Mol. Cell. Biol. 5:3251–3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466–6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072–2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32–39; Tratschin et al. (1984) J. Virol.51:611–619; and Flotte et al. (1993) J. Biol. Chem. 268:3781–3790).

The efficacy of a particular expression vector system and method ofintroducing nucleic acid into a cell can be assessed by standardapproaches routinely used in the art. For example, DNA introduced into acell can be detected by a filter hybridization technique (e.g., Southernblotting) and RNA produced by transcription of introduced DNA can bedetected, for example, by Northern blotting, RNase protection or reversetranscriptase-polymerase chain reaction (RT-PCR). The gene product canbe detected by an appropriate assay, for example by immunologicaldetection of a produced protein, such as with a specific antibody, or bya functional assay to detect a functional activity of the gene product.

A modulatory agent, such as a chemical compound that modulates theBCL-xγ anti-apoptotic activity, can be administered to a subject as apharmaceutical composition. Such compositions typically comprise themodulatory agent and a pharmaceutically acceptable carrier. As usedherein the term “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions. Pharmaceutical compositions can be prepared asdescribed herein.

There are a wide variety of pathological conditions for which BCL-xγmodulating agents of the present invention can be used in treatment. Inone embodiment, such agents modulate apoptosis in a cell. In a furtherembodiment this method can be used to treat a subject suffering from adisorder which would benefit from the modulation of apoptosis. In apreferred embodiment, BCL-xγ is modulated to enhance apoptosis of a Tcell, such as to promote the negative selection of autoreactive T cells.In another preferred embodiment, BCL-xγ is modulated to suppressapoptosis in a T cell, such as in the promotion of T cell survival inHIV infected T cells.

Other exemeplary disorders for which modulation of BCL-xγ can be used intreatment include, but are not limited to, various immune-mediateddisorders. The term disorder is meant to include both normal conditionsthat would benefit from an alteration in BCL-xγ activity and variousdisease states.

Since the subject BCL-xγ modulating agents can either increase ordecrease BCL-xγ activity, the agents will be useful for both stimulatingor suppressing immune responses.

In certain cases, the subject modulating agents can also be used toinhibit responses in clinical situations where it is desirable todownmodulate T cell survival. For example, it may be desirable todownmodulate BCL-xγ activity to promote T cell apoptosis, thus limitingT cell responsiveness in certain disorders. Examples include:graft-versus-host disease, cases of transplantation, and autoimmunediseases (including, for example, diabetes mellitus, arthritis(including rheumatoid arthritis, juvenile rheumatoid arthritis,osteoarthritis, psoriatic arthritis), multiple sclerosis, myastheniagravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis(including atopic dermatitis and eczematous dermatitis), psoriasis,Sjögren's Syndrome, including keratoconjunctivitis sicca secondary toSjögren's Syndrome, alopecia areata, allergic responses due to arthropodbite reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drugeruptions,leprosy reversal reactions, erythema nodosum leprosum,autoimmune uveitis, allergic encephalomyelitis, acute necrotizinghemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Crohn's disease, Graves ophthalmopathy, sarcoidosis,primary biliary cirrhosis, uveitis posterior, and interstitial lungfibrosis). Downmodulation of BCL-xγ will also be desirable in cases ofallergy such as, atopic allergy.

Conversely, it will be desirable to upregulate BCL-xγ activity to treatimmunodeficiency diseases, such as primary immunodeficiencies(including, severe combined immunodeficiency, adenosine deaminasedeficiency, purine nucleoside phosphorylase deficiency, MHC class IIdeficiency, reticular dysgenesis, X-linked agammaglobulinemia, X-linkedhypogammaglobulinemia, Ig deficiency with increased IgM, Ig heavychain-gene deletions, k-chain deficiency IgA deficiency, selectivedeficiency of IgG subclass, common variable immunodeficiency, transienthypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxiatelangiectasia, DiGeorge syndrome, Bloom syndrome, Fanconi anemia, andDown syndrome-related immunodeficiency, as well as other syndromesassociated with immunodeficiency) and immunodeficiencies resulting fromother causes, such as HIV disease/AIDS.

Additionally, it may be desirable to upregulate BCL-xγ activity toincrease T cell survival in the case of other disorders. For example,cellular responses to tumors, or pathogens, such as viruses, bacteria,fungi, parasites and the like, may be enhanced and/or prolonged, bypromoting T cell survival, thus enhancing T cell responses with thesubject modulating agents.

VI. Pharmaceutical Preparations

The subject modulating agents can be administered to a subject attherapeutically effective doses to treat or ameliorate a disorderbenefiting from the modulation of BCL-xγ. The data obtained from cellculture assays and animal studies can be used in formulating a range ofdosages for use in humans. The dosage of such modulating agents liespreferably within a range of circulating or tissue concentrations thatinclude the ED50 with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any modulating agent used in the method ofthe invention, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose may be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC50 (i.e., the concentration of the test modulating agent whichachieves a half-maximal inhibition of symptoms) as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans. Levels in plasma may be measured, for example,by high performance liquid chromatography.

In clinical settings, the gene delivery systems for the therapeuticbcl-xγ gene can be introduced into a patient by any of a number ofmethods, each of which is familiar in the art. For example, apharmaceutical preparation of the gene delivery system can be introducedsystemically, e.g., by intravenous injection, and specific transductionof the protein in the target cells occurs predominantly from specificityof transfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al.(1994) PNAS 91: 3054–3057). A mammalian bcl-xγ gene, such as representedin SEQ ID NO:1, or a sequence homologous thereto can be delivered in agene therapy construct by electroporation using techniques described,for example, by Dev et al. ((1994) Cancer Treat Rev 20:105–115).

The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery system can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

Pharmaceutical preparations for use in accordance with the presentinvention may also be formulated in conventional manner using one ormore physiologically acceptable carriers or excipients. Thus, themodulating agents and their physiologically acceptable salts andsolvates may be formulated for administration by, for example,injection, inhalation or insufflation (either through the mouth or thenose) or oral, buccal, parenteral or rectal administration.

For such therapy, the modulating agents of the invention can beformulated for a variety of loads of administration, including systemicand topical or localized administration. Techniques and formulationsgenerally may be found in Remmington's Pharmaceutical Sciences, MeadePublishing Co., Easton, Pa. For systemic administration, injection ispreferred, including intramuscular, intravenous, intraperitoneal, andsubcutaneous. For injection, the oligomers of the invention can beformulated in liquid solutions, preferably in physiologically compatiblebuffers such as Hank's solution or Ringer's solution. In addition, theoligomers may be formulated in solid form and redissolved or suspendedimmediately prior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical preparations may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated to give controlled release ofthe active modulating agent.

For administration by inhalation, the preparations for use according tothe present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the modulatingagent and a suitable powder base such as lactose or starch.

The modulating agents may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The modulating agents may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the modulatingagents may also be formulated as a depot preparation. Such long actingformulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the modulating agents may be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives. In addition, detergents may be used to facilitatepermeation. Transmucosal administration may be through nasal sprays orusing suppositories. For topical administration, the oligomers of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art.

The compositions may, if desired, be presented in a pack or dispenserdevice, or as a kit with instructions. The composition may contain oneor more unit dosage forms containing the active ingredient. The pack mayfor example comprise metal or plastic foil, such as a blister pack. Thepack or dispenser device may be accompanied by instructions foradministration.

VII. Transgenic Animals

The present invention also provides for transgenic animals in whichexpression of a genomic sequence or cDNA encoding a functional BCL-xγprotein is enhanced, induced, disrupted, prevented or suppressed. Thetransgenic animals produced in accordance with the present inventionwill include exogenous genetic material. As set out above, the exogenousgenetic material will, in certain embodiments, be a DNA sequence whichresults in the production of a BCL-xγ protein (either agonistic orantagonistic), an antisense transcript, or a BCL-xγ mutant. Further, insuch embodiments, the sequence will be attached to a transcriptionalcontrol element, e.g., a promoter, which preferably allows theexpression of the transgene product in a specific type of cell.

As used herein, the term “transgene” means a nucleic acid sequence(whether encoding or antisense to one of the mammalian BCL-xγ proteins),which is partly or entirely heterologous, i.e., foreign, to thetransgenic animal or cell into which it is introduced, or, is homologousto an endogenous gene of the transgenic animal or cell into which it isintroduced, but which is designed to be inserted, or is inserted, intothe animal's genome in such a way as to alter the genome of the cellinto which it is inserted (e.g., it is inserted at a location whichdiffers from that of the natural gene or its insertion results in aknockout). A transgene can include one or more transcriptionalregulatory sequences and any other nucleic acid, such as introns, thatmay be necessary for optimal expression of a selected nucleic acid.

A “transgenic animal” refers to any animal, preferably a non-humanmammal, bird or an amphibian, in which one or more of the cells of theanimal contain heterologous nucleic acid introduced by way of humanintervention, such as by transgenic techniques well known in the art.The nucleic acid is introduced into the cell, directly or indirectly byintroduction into a precursor of the cell, by way of deliberate geneticmanipulation, such as by microinjection or by infection with arecombinant virus. The term genetic manipulation does not includeclassical cross-breeding, or in vitro fertilization, but rather isdirected to the introduction of a recombinant DNA molecule. Thismolecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of one of the mammalian BCL-xγ proteins, e.g., either agonistic orantagonistic forms. However, transgenic animals in which the recombinantbcl-xγ gene is silent are also contemplated, as for example, the FLP orCRE recombinase dependent constructs described below. Moreover,“transgenic animal” also includes those recombinant animals in whichgene disruption of one or more bcl-xγ genes is caused by humanintervention, including both recombination and antisense techniques.

The “non-human animals” of the invention include mammals such asrodents, non-human primates, sheep, dog, cow, chickens, amphibians,reptiles, etc. Preferred non-human animals are selected from the rodentfamily including rat and mouse, most preferably mouse. The term“chimeric animal” is used herein to refer to animals in which therecombinant gene is found, or in which the recombinant is expressed insome but not all cells of the animal. The term “tissue-specific chimericanimal” indicates that one of the recombinant mammalian bcl-xγ genes ispresent and/or expressed or disrupted in some tissues but not others.

These systems may be used in a variety of applications. For example, thecell- and animal-based model systems may be used to further characterizebcl-xγ genes and proteins. In addition, such assays may be utilized aspart of screening strategies designed to identify modulating agentswhich are capable of ameliorating disease symptoms. Thus, the animal-and cell-based models may be used to identify drugs, pharmaceuticals,therapies and interventions which may be effective in treating disease.

One aspect of the present invention concerns transgenic animals whichare comprised of cells (of that animal) which contain a transgene of thepresent invention and which preferably (though optionally) express anexogenous BCL-xγ protein in one or more cells in the animal. A bcl-xγtransgene can encode the wild-type form of the protein, or can encodehomologs thereof, including both agonists and antagonists, as well asantisense constructs. In preferred embodiments, the expression of thetransgene is restricted to specific subsets of cells, tissues ordevelopmental stages utilizing, for example, cis-acting sequences thatcontrol expression in the desired pattern. In the present invention,such mosaic expression of a BCL-xγ protein can be essential for manyforms of lineage analysis and can additionally provide a means to assessthe effects of, for example, lack of BCL-xγ expression which mightgrossly alter development in small patches of tissue within an otherwisenormal embryo. Toward this end, tissue-specific regulatory sequences andconditional regulatory sequences can be used to control expression ofthe transgene in certain spatial patterns. Moreover, temporal patternsof expression can be provided by, for example, conditional recombinationsystems or prokaryotic transcriptional regulatory sequences.

Genetic techniques which allow for the expression of transgenes can beregulated via site-specific genetic manipulation in vivo are known tothose skilled in the art. For instance, genetic systems are availablewhich allow for the regulated expression of a recombinase that catalyzesthe genetic recombination a target sequence. As used herein, the phrase“target sequence” refers to a nucleotide sequence that is geneticallyrecombined by a recombinase. The target sequence is flanked byrecombinase recognition sequences and is generally either excised orinverted in cells expressing recombinase activity. Recombinase catalyzedrecombination events can be designed such that recombination of thetarget sequence results in either the activation or repression ofexpression of one of the subject BCL-xγ proteins. For example, excisionof a target sequence which interferes with the expression of arecombinant bcl-xγ gene, such as one which encodes an antagonistichomolog or an antisense transcript, can be designed to activateexpression of that gene. This interference with expression of theprotein can result from a variety of mechanisms, such as spatialseparation of the bcl-xγ gene from the promoter element or an internalstop codon. Moreover, the transgene can be made wherein the codingsequence of the gene is flanked by recombinase recognition sequences andis initially transfected into cells in a 3′ to 5′ orientation withrespect to the promoter element. In such an instance, inversion of thetarget sequence will reorient the subject gene by placing the 5′ end ofthe coding sequence in an orientation with respect to the promoterelement which allow for promoter driven transcriptional activation.

The transgenic animals of the present invention all include within aplurality of their cells a transgene of the present invention, whichtransgene alters the phenotype of the “host cell” with respect toregulation of cell growth, death and/or differentiation. Since it ispossible to produce transgenic organisms of the invention utilizing oneor more of the transgene constructs described herein, a generaldescription will be given of the production of transgenic organisms byreferring generally to exogenous genetic material. This generaldescription can be adapted by those skilled in the art in order toincorporate specific transgene sequences into organisms utilizing themethods and materials described below.

In an illustrative embodiment, either the cre/loxP recombinase system ofbacteriophage P1 (Lakso et al. (1992) PNAS 89:6232–6236; Orban et al.(1992) PNAS 89:6861–6865) or the FLP recombinase system of Saccharomycescerevisiae (O'Gorman et al. (1991) Science 251:1351–1355; PCTpublication WO 92/15694) can be used to generate in vivo site-specificgenetic recombination systems.

Accordingly, genetic recombination of the target sequence is dependenton expression of the Cre recombinase. Expression of the recombinase canbe regulated by promoter elements which are subject to regulatorycontrol, e.g., tissue-specific, developmental stage-specific, inducibleor repressible by externally added agents. This regulated control willresult in genetic recombination of the target sequence only in cellswhere recombinase expression is mediated by the promoter element. Thus,the activation expression of a recombinant BCL-xγ protein can beregulated via control of recombinase expression.

Use of the cre/loxP recombinase system to regulate expression of arecombinant BCL-xγ protein requires the construction of a transgenicanimal containing transgenes encoding both the Cre recombinase and thesubject protein. Animals containing both the Cre recombinase and arecombinant bcl-xγ gene can be provided through the construction of“double” transgenic animals. A convenient method for providing suchanimals is to mate two transgenic animals each containing a transgene,e.g., a bcl-xγ gene and recombinase gene.

One advantage derived from initially constructing transgenic animalscontaining a bcl-xγ transgene in a recombinase-mediated expressibleformat derives from the likelihood that the subject protein, whetheragonistic or antagonistic, can be deleterious upon expression in thetransgenic animal. In such an instance, a founder population, in whichthe subject transgene is silent in all tissues, can be propagated andmaintained. Individuals of this founder population can be crossed withanimals expressing the recombinase in, for example, one or more tissuesand/or a desired temporal pattern. Thus, the creation of a founderpopulation in which, for example, an antagonistic bcl-xγ transgene issilent will allow the study of progeny from that founder in whichdisruption of BCL-xγ mediated induction in a particular tissue or atcertain developmental stages would result in, for example, a lethalphenotype.

Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the bcl-xγ transgene.Exemplary promoters and the corresponding trans-activating prokaryoticproteins are given in U.S. Pat. No. 4,833,080.

Moreover, expression of the conditional transgenes can be induced bygene therapy-like methods wherein a gene encoding the trans-activatingprotein, e.g. a recombinase or a prokaryotic protein, is delivered tothe tissue and caused to be expressed, such as in a cell-type specificmanner. By this method, a bcl-xγ transgene could remain silent intoadulthood until “turned on” by the introduction of the trans-activator.

In one embodiment, gene targeting, which is a method of using homologousrecombination to modify an animal's genome, can be used to introducechanges into cultured embryonic stem cells. By targeting a bcl-xγ geneof interest e.g., in embryonic stem (ES) cells, these changes can beintroduced into the germlines of animals to generate chimeras. The genetargeting procedure is accomplished by introducing into tissue culturecells a DNA targeting construct that includes a segment homologous to atarget bcl-xγ locus, and which also includes an intended sequencemodification to the bcl-xγ genomic sequence (e.g., insertion, deletion,point mutation). The treated cells are then screened for accuratetargeting to identify and isolate those which have been properlytargeted.

Methods of culturing cells and preparation of knock out constructs forinsertion are known to the skilled artisan, such as those set forth byRobertson in: Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. IRL Press, Washington, D.C. [1987]); byBradley et al. (1986) Current Topics in Devel. Biol. 20:357–371); and byHogan et al. (Manipulating the Mouse Embryo: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1986]).

Introduction of the transgenic constructs nucleotide sequence into theembryo may be accomplished by any means known in the art such as, forexample, microinjection, electroporation, calcium phosphate, orlipofection. Retroviral infection can also be used to introducetransgene into a non-human animal. The developing non-human embryo canbe cultured in vitro to the blastocyst stage. During this time, theblastomeres can be targets for retroviral infection (Jaenich, R. (1976)PNAS 73:1260–1264).

Other methods of making knock-out or disruption transgenic animals arealso generally known. See, for example, Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).Recombinase dependent knockouts can also be generated, e.g. byhomologous recombination to insert target sequences, such that tissuespecific and/or temporal control of inactivation of a bcl-xγ-gene can becontrolled by recombinase sequences.

Animals containing more than one knockout construct and/or more than onetransgene expression construct are prepared in any of several ways. Apreferred manner of preparation is to generate a series of mammals, eachcontaining one of the desired transgenic phenotypes. Such animals arebred together through a series of crosses, backcrosses and selections,to ultimately generate a single animal containing all desired knockoutconstructs and/or expression constructs, where the animal is otherwisecongenic (genetically identical) to the wild type except for thepresence of the knockout construct(s) and/or transgene(s).

The contents of all cited references, including literature references,issued patents, published patent applications as cited throughout thisapplication are hereby expressly incorporated by reference. The practiceof the present invention will employ, unless otherwise indicated,conventional techniques of cell biology, cell culture, molecularbiology, transgenic biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds 1984); Transcription And Translation (B. D.Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154and 155 (Wu et al. eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); HandbookOf Experimental Immunology, Volumes I–IV (D. M. Weir and C. C.Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

EXEMPLIFICATION

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way.

Example 1 Cloning of the BCL-x γ Gene from a T-Cell-Derived DNA Library

A thymus λ ZapII cDNA library derived from BALB/cJ mice (Stratagene, LaJolla Calif.) was screened with a ³²P-labeled 60 mer oligonucleotide(5′-GGGGTGATGTGGAGCTGGGATGTCAGGTCACTGAATGCCCGCCGGTACCGCA GTTCAAAC-3′,SEQ ID NO:5) derived from a human bcl-x cDNA sequence (base pairs423–483) homologous to the region of chicken bcl-x (Boise et al. 1993Cell 74, 597–608) by hybridization to approximately 10⁶ phages blottedon 20 filters in duplicates according to a modification of the protocolsby Wood et al. (1985. Proc. Natl. Acad. Sci. USA 82:1585) and Jacobs etal. (1988 Nucleic Acids Res. 16, 4637–4650). Briefly, the filters wereprehybridized for 2 hours and hybridized overnight in 6×NaCl/Cit, 5×Denhardt's solution containing boiled sonicated salmon sperm DNA at 0.1mg/ml at 42° C. The filters were rinsed three times with 6×NaCl/Cit at4° C. and washed twice for 30 mm with 6×NaCl/Cit at 4° C. The filterswere then rinsed with th Me₄NCl (tetramethylammonium chloride) washsolution including 50 mM Tris-Cl, pH 8.0, 2 mM EDTA and 0.1% SDS at 37°C., 45° C., 50° C., 55° C., 60° C., 65° C. and 70° C. respectively for20 mm at each temperature. Six clones were isolated and purified (Bclx5,6,7,8,10,11); four contained inserts of the same length and sequencedand corresponded to Bcl-xL cDNA and a fifth to Bcl-xβ (GenBank accessionnumbers U51279, U51278) (M. Gonzalez-Garcia, et al., Development 120,3033 (1994), W. Fang, J. J. Rivard, D. L. Mueller, T. W. Bebrens, J.Immunol. 153, 4388 (1994). Sequencing was done by double-stranded DNAdideoxy chain termination method using T7 DNA polymerase (US Biochem).Sequencing was performed twice on both strands by walking along thecDNAs with primers custom-synthesized by Amitof (Cambridge, Mass.).Other DNA manipulations were performed according to standard protocols(Sambrook et al., 1989 Molecular Cloning. A laboratory manual 2ndedition. Cold Spring Harbour Laboratory Press).

One clone, termed Bclx7 (GenBank accession number U51277), contained a1384 bp insert comprised of a 5′ noncoding region of 377 nucleotides, anORF of 708 nucleotides and a 3′ noncoding region of 299 nucleotides(FIG. 1A). This represented a novel isoform of the Bcl-x gene in whichthe 3′ region of Bcl-xL was replaced by a 144 bp sequence which predictsa unique C-terminus of 47 amino acids (FIG. 1A). This insert did notrepresent a cloning artifact because the novel 144 bp subsequence beginsprecisely at a conserved donor/acceptor splice site used by murine andhuman Bcl-x isoforms (L. H. Boise et al., Cell 74, 597 (1993), M.Gonzalez-Garcia, et al., Development 120, 3033 (1994), W. Fang, J. J.Rivard, D. L. Mueller, T. W. Behrens, J. Immunol. 153, 4388 (1994) (FIG.1B) and the sequence was independently cloned from thymocyte RNA (usinga primer specific for a conserved region of murine Bcl-x and a Bcl-x-7specific primer. The recovered thymic sequence was identical to the cDNAinsert isolated from the λ Zap II cDNA library and the new isoform wasdesignated bcl-xγ.

Example 2. PCR Cloning of the bcl-xγ Gene

Total RNA was extracted from murine thymus (BALB/c) after homogenizationwith a Brinkmann homogenizer (Brinkmann Instruments, New York). Reversetranscription/PCR was performed on a GeneAmp PCR 9600 (Perkin-Elmer)using a GeneAmp RNA-PCR kit (Perkin-Elmer Cetus) typically at 42° C. for30 min, 99° C. for 5 min and 4° C. for 5 min, according to themanufacturer's protocols. All oligonucleotide primers were synthesizedby Amitof (Cambridge Mass.). For PCR cloning, one primer specific forthe 5′ upstream bcl-x common region(5′-TCGCTCGCCCACATCCCAGCTTCACATAACCCC-3′, SEQ ID NO:6) (Note:underlining in original) and a second primer specific for the 3′downstream bcl-xγ region (5′-CTGGTTCGGCCCACGTCCTTCCTGAAGT-CCTCC-3′, SEQID NO:7) (Note: underlining in original) were used (underliningindicates regions specific for PCR-Direct™ cloning kit [Clontech]).Amplification products were separated on agarose gels, purified withGeneclean II kit (Bio101), subcloned into the PCR-Direct™ vector andsequenced by the chain termination method.

Example 3 In Vitro Transcription and Translation of bcl-xγ

In vitro transcription and translation assays using linearizedrecombinant bcl-x plasmids were performed to confirm the length of theORF deduced from the cDNA sequence of bcl-xγ (FIG. 2). Recombinantplasmid bluescripts containing cDNAs from bcl-xL, bcl-xS, bcl-x-β andbcl-xγ were linearized with a unique PstI restriction enzyme at the 3′end of the insert and polycloning sites of plasmid. In vitrotranscription and translation, using the linearized recombinantbluescript as template, were performed using a TNT T7/T3-coupledreticulocyte lysate system, according to the manufacturer's protocol(Promega). Briefly, 1 μg of linearized plasmid in which T7 promotersequence was located upstream of the cDNA insert of bcl-x isoforms wasadded into 50 μl of TNT reticulocyte lysate supplemented with T7 RNApolymerase, RNAase inhibitor, ³⁵S-methionine, and a mixture of otheramino acids. After incubation for 90 min at 30° C., 10 μl of eachnewly-synthesized ³⁵S-methionine labeled protein were analyzed by a 12%SDS polyacrylamide gel electrophoresis and autoradiography. The apparentsize of BCL-xγ protein after in vitro transcription/translation isconsistent with the size predicted from its open reading frame, sincethe BCL-xγ protein product migrates at a position similar to BCL-xL (233a.a. residues) (FIG. 1B) and more slowly than the BCL-xβ protein (209a.a.) and the BCL-xS (170 a.a.). Molecular weight standards (kDa) areindicated on the left margin of the figure.

As expected from the predicted bcl-xγ ORF of 708 nt/235 aa, the apparentsize of the translated Bcl-xγ protein was slightly larger than the (233aa) BCL-xL protein product and considerably larger than both the (170aa) Bcl-xS (W. Fang, J. J. Rivard, D. L. Mueller, T. W. Bebrens, J.Immunol. 153, 4388 (1994) and the (209 aa) BCL-xβ proteins (M.Gonzalez-Garcia, et al., Development 120, 3033 (1994). Analysis of thehydrophobicity of the unique C-terminus of the BCL-xγ protein indicatedthat BCL-xγ lacks an obvious hydrophobic domain flanked by chargedresidues (FIG. 1C), which are present in human and murine BCL-xL andBCL-xS (L. H. Boise et al., Cell 74, 597 (1993), D. Hockenberry, G.Nunez, C. Milliman, R. D. Schreiber, S. J. Korsmeyer, Nature 348, 334(1990); M. Nguyen, D. G. Millar, V. W. Yong, S. J. Korsmeyer, G. C.Shore, . J. Biol. Chem. 268, 25265 (1993). Hydrophobicity of BCL-xL andBCL-xγ was calculated using the GCG program based on Goldman's (solidline) or Kyte-Doolittle's (dashed line) algorithm. A 33 aa region withinthe C-terminal domain of BCL-xγ showed strong homology with theconsensus sequence of ankyrin-like domains (FIG. 1D, the consensussequence of ankyrin-like domain which spans 33 amino acid residues indifferent species is shown) that are embedded in a number ofintracellular proteins including BCL-3, which uses this subsequence tobind to NF-κB p50 (H. N. Hatada et al., Proc. Natl. Acad. Sci. USA 89,2489 (1992).

Example 4 Gene Expression in Prokaryotic Cells as Fusion Protein

BCL-xγ has been successfully expressed in E. coli. The bcl-xγ cDNA wasamplified by PCR using primer 5′-CCGGGAATTCATCTCAGAGCAACCGGGAGCTGGTG-3′(SEQ ID NO:8), specific for the BCL-x common region, and a second primer5′-CCAGGAATTCGGATCCCGTCCTTCCTGAAGTCCTCCT-3′, (SEQ ID NO:9), specific forthe unique region of bcl-xγ, which contain an EcoRi endonucleaserestriction site, respectively. These primers flank the 5′ and 3′ endsof the full mature bcl-xγ open reading frame. The amplified DNA has beenpurified, cut with EcoRI and ligated into the EcoRI site of pGEX-3X, ahigh expression prokaryotic vector and E. coli DH5α strain has beentransformed. Ampicillin resistant colonies were screened for thesynthesis of GST-BCL-x γ fusion protein by SDS-PAGE. Recombinant proteinwas purified from bacterial lysate by affinity chromatography onglutathione-agarose resin.

Example 5 Measurement of Gene Expression by RT/PCR

In addition to the bcl-xγ specific primer set, a primer containing a 3′unique region of bcl-xL and bcl-xS (5′-CCACCAACAAGACAGGCT-3′, SEQ IDNO:10) was used to pair with the 5′ primer from the bcl-x common regionfor amplification of the bcl-xL fragment. Similarly, a primer calledDTM1 (5′-CTCTCCTCCCTCACACACCCCTCTC-3′, SEQ ID NO:11) complementary tothe 3′ specific region of Bcl-xΔTM and a primer called 3ep (5′AAGATACAGGTCCCTTAAA-3′, SEQ ID NO:12) complementary to the 3′ specificregion of bcl-xβ were used to pair with the 5′ primer from the bcl-xcommon region for amplification of bcl-xΔTM and bcl-xβ. A pair ofprimers specific for mouse β-actin (5′-ATGGATGACGATATCGCTGC-3′(SEQ IDNO:13) and 5′-CTAGAAGCACTTGCGGTGCAC-3′, SEQ ID NO:14) was used as aninternal control for RT-PCR to evaluate usage of comparable amounts ofcDNA in all samples. Furthermore, in activated O3 clones primers forinterleukin-2 (5′-TTCAAGCTCCACTTCAAGCTC-3′(SEQ ID NO:15) and5′-GACAGAAGGCTATCCATCTCC-3′, SEQ ID NO:16) and interferon-γ(5′-TGCATCTTGGCTTTGCAGCTCTTCCTCATG-3′(SEQ ID NO:17) and5′-TGGACCTGTGGGTTGTTGACCTCAAACTTG-3′, SEQ ID NO:18) served as controlsfor efficient stimulation. PCR reactions were typically performedthrough 35–45 cycles using Taq DNA polymerase (Perkin-Elmer)supplemented with TaqStart antibody in order to maintain the specificityof amplified fragments (Clontech). Each 3-step thermal cycle consistedof 30 seconds at 94° C., 30 seconds at 60° C., 30 seconds at 72° C. Toencompass the exponential phase of the amplification, 25 μl of thereaction mix was removed at regular intervals during PCR, as previouslydescribed (Moore et al., 1994 Immunology 81, 115–119). A negativecontrol containing all reagents except cDNA was included in each PCRanalysis (Moore et al., 1995 J. Immunol. 155, 4653–4660). PCR fragmentsin 10 μl of each sample were visualized by agarose gel electrophoresisand ethidium bromide staining and were positively identified by size,bcl-xγ fragments were further confirmed by Southern blot hybridization.The RT-PCR products amplified with bcl-xγ-specific primers wereseparated on agarose gels and blotted onto a nylon filter (MicronSeparations Inc.) via upward capillary transfer in 20×SSC before filterswere air-dried and subjected to UV-crosslinking.

Example 6 Expression of bcl-xγ

According to RT-PCR, bcl-xL (as well as bcl-xβ and bcl-xΔTM) isexpressed in all tissues tested, including brain, eyes, heart,intestine, kidney, liver, lung, lymph nodes, and thymus (FIG. 3A),consistent with previous reports (L. H. Boise et al., Cell 74, 597(1993), M. Gonzalez-Garcia, et al., Development 120, 3033 (1994).Products were analyzed on 1% agarose gels stained with ethidium bromide.Molecular weight markers are indicated on the right margin of the gels.In contrast, bcl-xγ expression was detected in thymus, lymph node, lungand eye, but not brain, heart, intestine, kidney, liver (FIG. 3A). Thespecificity of the amplified bcl-xγ fragments in these tissues wasconfirmed by Southern blotting with a bcl-xγ-specific probe (FIG. 3A).The RT-PCR products amplified with bcl-xγ-specific primers wereseparated on agarose gels and blotted onto a nylon filter (MicronSeparations Inc.) and subjected to UV-crosslinking. A 360 bpbcl-xγ-specific probe prepared by PCR amplification using therecombinant plasmid bclx7 encoding bcl-xγ as a template and usingprimers that do not overlap with the primers was used to detect geneexpression followed by labeling with [α-³²P]dCTP (3000 Ci/mmol, NEN) byrandom oligomer priming (Oligolabeling kit, Pharmacia). The upstream(5′-GGTGTGAGTGGAGGTACA-3′, SEQ ID NO:23) and downstream(5′-CCCCTCTGTTGATTTTCTG-3′, SEQ ID NO:24) primers were used as probes.Radiolabeled probes were purified using Nick-spin columns (Pharmacia) toremove excess unincorporated radioactive nucleotides before used forhybridization overnight at 42° C. in 6×SSC buffer containing 50%formamide. The filters were washed in 2×SSC containing 0.1% SDS at 42°C. for 30 min. and in 0.2×SSC containing 0.1% SDS at 65° C. for 30 min.followed by autoradiography.

Failure to detect bcl-xγ in tissues such as brain, heart, intestine,kidney and liver by RT-PCR did not result from degraded preparations ofRNA from these tissues since the ratio of ethidium bromide-stained 28SrRNA to 18S rRNA bands in agarose gels was the same for all tissues andbcl-xL, β-actin and other genes were successfully amplified by RT-PCRfrom the same RNA samples which were negative for bcl-xγ. These resultsindicate that expression of the bcl-xγ isoform is more restricted thanother members of the bcl-x family.

Expression of bcl-xγ was tested in T-cells, B-cells or monocytes. bcl-xγwas not expressed in peripheral lymphoid tissues of Rag-2 deficientmice, consistent with its selective expression in lymphocytes.Furthermore, bcl-xγ was expressed in lymph nodes of BALB/c but notBALB/c nu/nu mice, suggesting that its expression is confined toT-lymphocytes (FIG. 3B). The analysis of cDNA from lymph nodes (LN) ofnormal, BALB/c nu/nu and Rag-2^(−/−) mice indicated that bcl-x γ isamplified in LN of normal, but not nu/nu donors (PCR amplification ofβ-actin fragment served as control.

Additional analysis of bcl-xγ expression in the thymus indicated that itis not detectable in double negative (DN) cells from normal orrecombinase-activating gene (RAG)-2 deficient (RAG-2^(−/−)) donors, norin thymocytes from mice which are deficient in TCR-β chain and fail toundergo TCR-dependent maturation into double positive (DP) thymocytes.bcl-xγ is expressed by double positive (DP) thymocytes, sincepreparations that contained approximately 90% double positive (DP) and10% single positive (SP) cells expressed bcl-xγ while purified SPthymocytes did not express detectable bcl-γ.

Bcl-xγ expression in DP thymocytes depends on engagement of the TCR byMHC/peptide ligands in the thymus, since bcl-xγ was not detectable inthymocytes from mutant mice deficient in MHC class I/II (MHCdouble-deficient mice) (FIG. 3C). By contrast, expression of bcl-xL andbcl-xS (and bcl-xβ, -ΔTM, not shown) was unchanged in thymocytes fromboth MHC double-deficient mice and TCR-β deficient mice, suggesting thatexpression of other bcl-x isoforms in the thymus does not depend on TCRligation.

Expression of bcl-xγ in the thymuses of normal, cortisone-treated andmutant mice was also detected by RT-PCR. For cortisone treatment ofmice, 2.5 mg/mouse of Cortisone Acetate (Merck Sharp and Dohme, U.S.A.)was injected i.p. into one-month old C57BL/6J mice 48 hours beforeanimals were sacrificed as described previously [R. Scollay, K.Shortman, Thymus 5, 245 (1983)]. PCR analysis showed that expression ofMHC class I and II, TCR-α, and Fas genes are not required for expressionof bcl-xL and bcl-xS since in the thymuses from all of those mutant micebcl-xL and bcl-xS were expressed in a comparable level. On the contrary,bcl-xγ was only expressed in the thymuses from normal mice, TCR-αknock-out mice and Fas gene mutant mice. The specificity of bcl-xγfragments amplified in RT-PCR was confirmed by a Southern blothybridization using a [32-P] dCTP-labeled bcl-xγ-specific probe whichdid not overlap with either primer used in RT-PCR. (FIG. 3C).

Example 7 Sequence Variations within the 3′ Noncoding Region of bcl-xγ

bcl-x γ is expressed differently in different murine tissues. The twoobserved sizes of bcl-x γ reflect nucleotide insertions within the 3′non-coding region according to cloning/DNA sequencing. The length andcontent of the 3′ noncoding region may affect mRNA translationalefficiency or stability (Tanguay and Gallic, 1996 Mol. Cell. Biol. 16,146–156).

Example 8 Association of BCL-xγ with the T Cell Receptor

BCL-xγ expression was tested in a variety of cell types. O3 is a murineCD4+Th1 clone derived from BALB/c mice after in vitro selection forproliferation to OVA in association with presenting cells (APC) ofBALB/c mice [S. Friedman, D. Sillcocks, H. Cantor, Immuogenetics 26, 193(1987)]. The AF3.G7 hybridoma, generated by fusing cow insulin-immuneC57BL/6 lymph node cells with the BW5147 thymoma line, expresses aV_(β)6⁺/V_(α)3.2⁺ TCR and responds to both cow insulin peptide and toMTV-7 according to IL-2 production [D. G. Spinella et al., J. Immunol.138, 3991 (1987)]. EL4 is a mouse lymphoma cell line established inC57BL/6N mice which produces high titers of murine IL-2[J. Wein, E.Roberts, Cancer Res. 25, 1753 (1965)]. Bcl-xγ expression was notdetectable in the resting murine T_(H)1 clone O3, but increasedsubstantially by 4 hours after CD3 ligation (MM6). Bcl-xγ was notexpressed after IL-2 activation of these cells, although T-cell [³H]-TdRincorporation after IL-2 activation or CD3 ligation was similar (FIG.4A,B). After exposure of O3 T-cells to plate-bound anti-CD3 antibody forthe indicated intervals, total RNA was extracted and RT-PCRamplification by an interleukin-2 and interferon-γ fragment indicatedactivation as early as 4 hours. The specificity of BCL-xγ fragmentsamplified in RT-PCR was confirmed by a Southern blot hybridization usinga [³²-P]dCTP-labeled BCL-xγ specific probe which did not overlap witheither primer used in RT-PCR as described in FIG. 3A and wasdeliberately overexposed to detect weak hybridization in negative PCRlanes (negative groups). Primers specific for interleukin-2 andinterferon-γ were used as positive controls for T cell activation inRT-PCR. Oligonucleotides used as primers for PCR amplification of themouse interleukin-2 fragment were 5′-TTCAAGCTCCACTTCAAGCTC-3′ (SEQ IDNO:15) and 5′-GACAGAAGGCTATCCATCTCC-3′ (SEQ ID NO:16). Primer sequencesfor PCR amplification of the mouse interferon-γ fragment were5′-TGCATCTTGGCTTTGCAGCTCTTCCTCATG-3′, SEQ ID NO:17 and5′-TGGACCTGTGGGT-TGTTGACCTCAAACTTG-3′, SEQ ID NO:18. PCR-amplifiedfragments were analyzed on agarose gels followed by scanning andquantitation using an IS-1000 digital imaging system (Alpha InnotechCorp.), adjusting for exposure times so that the intensity of DNAfragment signals corresponded to the linear range of densitometricdetection. To ensure that comparisons of cDNA levels in differentsamples were based upon the same amount of cDNA in each sample, the areaunder the densitometric peak of each sample was divided by the areaunder the β-actin densitometric peak for the corresponding sample. Theratios of bcl-x isoforms and controls (IL-2, IFN-γ) cDNA to β-actin cDNAfor each sample are shown in Relative Densitometric Units.

Bcl-xγ expression after IL-2 stimulation was not detectable even afterthe number of PCR cycles was increased to the maximal number (50 cycles)before polymerase activity becomes limiting (D. M. Coen, In: CurrentProtocols in Molecular Biology. Ed. Ausubel, et al. Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., Volume 2, Chapter 15:15.01 (1994). In contrast, all other murine Bcl-x isoforms (bcl-xL, -β,-ΔTM, -S) were expressed in resting O3 cells and displayed similarincrements 8 hours after either IL-2R or CD3 ligation (FIGS. 4A,B).After incubation of O3 T-cells with 25 U/ml IL-2 resulting in levels of[³H]-thymidine incorporation that were similar to that obtained afterCD3 ligation, total RNA was extracted. These results show that BCL-xL,BCL-xβ and BCL-xΔTM are upregulated 4–24 hours after IL-2 treatment, butBCL-x γ expression is not detectable. The PCR amplified fragments onagarose gels were scanned, quantitated using the IS-1000 digital imagingsystem (Alpha Innotech Corp.) and normalized as described above. Theresults indicate that signaling through IL-2 receptor does notupregulate the expression of BCL-x γ. TCR-dependent expression of Bcl-xγwas not limited to non-transformed primary T-cell clones: neither theAF3.G7 insulin-specific T-cell hybridoma nor the EL4 lymphoma cell lineexpressed bcl-xγ unless CD3 was ligated, in contrast to all other bcl-xisoforms displayed constitutive expression in these cells which was notincreased after TCR ligation.

Example 9 Effect on T Cell Apoptosis

Since studies of previously described BCL-x isoforms have indicated thatthey either enhance (L. H. Boise et al., Cell 74, 597 (1993), M. F.Clarke, et al., Proc. Natl. Acad. Sci. USA, 92 11024 (1995); A. J. Minnet al., J. Biol. Chem. 271, 6306 (1996) or inhibit (L. H. Boise et al.,Cell 74, 597 (1993), M. Gonzalez-Garcia, et al., Development 120, 3033(1994), W. Fang, J. J. Rivard, D. L. Mueller, T. W. Behrens, J. Immunol.153, 4388 (1994), M. Gonzalez-Garcia, et al., Proc. Natl. Acad Sci. USA92, 4304 (1995) apoptosis, the effect of stable Bcl-xγ expression onapoptosis following TCR ligation was tested. A stable cell lineexpressing BCL-x γ was constructed. The plasmid pRC/RSV containingenhancer-promoter sequences from the Rous sarcoma virus long terminalrepeat (Invitrogen, San Diego Calif.) was used to constructpRC/RSV-Bcl-xL by inserting a 0.75 kb fragment which contained afull-length open reading frame of Bcl-xL. The pRC/RSV-bcl-xγ vector wasconstructed by inserting a 1.0 kb fragment containing the full-lengthORF of bcl-xγ. Correct orientation of bcl-xL and bcl-xγ inserts in therecombinant vector was confirmed by restriction enzyme digestion and DNAsequencing. Stable expression of the CTLL-2 T cell line (a mouse T-cellline derived from C57BL/6 H. E. Broome, C. M. Dargan, S. Krajewski, J.C. Reed, 1995. J. Immunol. 155, 2311). was achieved after transfectionin a 5×10⁶/0.5 ml with 10 μg Xbal-linearized vector by electroporationin a Gene-Pulser II (BioRad, CA) at 270 Volts and 950 μF for 20 msec.Two days after transfection, T-cells were diluted into 96-well plates at5×10³/0.1 ml or 1×10⁴/0.1 ml/well in media containing 750 μg/ml of G418and after two weeks, individual clones resistant to G418 were selected,expanded and maintained in medium containing 250 μg/ml of G418. Inaddition, the empty vector pRC/RSV was used to simultaneously transfectand expand CTLL-2 cells according to the same protocol. RT-PCR wasperformed to confirm efficient expression of transfected bcl-x genes inthe transfected clones using RNA and digested with RNAse-free DNAsebefore RT-PCR to avoid contamination in RNA preparations. cDNAs reversedtranscribed from total RNAs from these transfectants and cellstransfected with the pRC/RSV control vector was amplified with a vectorspecific primer paired with either a bcl-xL specific primer or a bcl-xγspecific primer, run on agarose gels and confirmed by Southern blothybridization with a ³²P-labeled DNA probe prepared from the cDNA codingfor the Bcl-x common region.

CD3 was ligated on CTLL-2 cells which stably overexpressed BCL-xγ(pRC/RSV-Bcl-xγ), BCL-xL (pRC/RSV-Bcl-xL) or a vector control (pRC/RSV).This led to apoptosis in 70% of the vector control transfectant cells,10% of transfectants which overexpressed Bcl-xγ and 13% of transfectantswhich overexpressed Bcl-xL (FIG. 5). Plates precoated with anti-mouseCD3 antibody (Pharmingen, San Diego Calif.) (5 μml) were washed threetimes before addition of CTLL-2 clones that had been rinsed 3× with theIL-2-free RPMI 1640 medium supplemented with 5% FCS and incubated at 2ml/well at a concentration of 1.25×10⁵/ml at 37° C. for 24 hrs.Incubation medium was replaced with fresh RPMI 1640 medium supplementedwith 5% FCS at 6 and 12 hours after plating cells to reduce secondaryresponses to potential growth factors secreted by cells activated afterTCR ligation. The percentage of cells undergoing apoptosis for eachtransfected clones was analyzed by propidium iodide (PI) staining [A. J.McGahon et al., Meth. Cell Biol. 46, 153 (1995)]; H. E. Broome, C. M.Dargan, S. Krajewsky, J. C. Reed, J. Immunol. 155, 2311 (1995)].Briefly, 24 hrs. after activation by plate-bound anti-CD3, cells wereharvested, rinsed twice with cold PBS containing 5 mM EDTA, fixed with50% ethanol in PBS containing 5 mM EDTA for 30 min at room temperatureand treated with 40 μg/ml of DNAse-free RNAse A in PBS for 30 min andstained with 50 μg/ml of propidium iodide in PBS for 30 minutes beforeanalysis in an Epics XL flow cytometry system using a standard settingof FL2 in semi-log mode (Coulter Inc.). Since partial loss of DNA fromapoptotic cells due to activation of endogenous endonuclease(s) and/ormarked condensation of the chromatin accompanies apoptosis and rendersthese areas of DNA inaccessible to PI staining, subdiploid cells withDNA concentrations lower than that of G0/G1 cells, were considered to beapoptotic [A. J. McGahon et al., Meth. Cell Biol. 46, 153 (1995)], whilecells in G0/G1, S, G2/M phases were scored as viable. The cell cycleprofile of CTLL-2 cells which stable express the indicated constructsafter activation by plate-bound anti-CD3. The distribution of cellsbetween the G1, S and G2/M phases of the cell cycle are shown; theabscissa indicates the relative cell number and the ordinate indicatesDNA content based on PI staining of pRC vector-transfected cells,BCL-xL-transfected cells, and BCL-x γ-transfected cells. The numbers inthe upper left comer represents the percent of cells which displayapparent DNA contents of less than diploid (subdiploid), correspondingto the subpopulation of apoptotic cells. These stable trasfectantsexpressed similar levels of CD3 according to immunofluoresence and allhad similar baseline levels of apoptosis (4–8%). These results arerepresentative of three experiments.

These experiments, and previous transfection/overexpression studies, donot define the physiological role of endogenous Bcl-x expression in TCR-dependent apoptosis. Activated T-cells (including T_(H)1 clone O3)undergoing apoptosis after CD3 ligation stain intensely and specificallywith Hoechst 33342 dye within 4–8 hours after activation, while theHoechst-negative subpopulation of activated T-cells goes on to divideand produce cytokines (Weber et al. Immunity 2:363, 1995). O3 T cellclones (1×10⁶/ml) were cultured on plates pre-coated with anti-CD3-ε (5μg/ml in PBS [_(p)H 8.5], Pharmingen; preincubated (37° C.) overnight)and incubated (37° C.) in DMEM plus 5% FBS before staining of activatedO3 T cells with Hoechst 33342 dye and propidium iodide before analysisby flow cytometry, as described (M. G. Ormerod et al., Cytometry 14, 595(1993). After gating out dead cells, activated T cell blasts were sortedinto Hoechst-negative (non-apoptotic) and Hoechst-positive (apoptotic)subpopulations on a Becton-Dickinson FACS (G. F. Weber, S.Ambromson-Leeman, H. Contor, Immunity 2, 363 (1995). Activated O3 cellswere analyzed for Bcl-x isoform expression after sorting intoHoechst-positive and Hoechst-negative fractions 5 hours after CD3ligation. Bcl-xγ was strongly expressed in the successfully-activatedHoechst-negative fraction, but was not detectable in theHoechst-positive fraction destined to undergo apoptosis (FIG. 6), evenafter maximum runs of 50 PCR cycles (D. M. Coen, In: Current Protocolsin Molecular Biology. Ed. Ausubel, et al. Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., Volume 2, Chapter 15: 15.01 (1994). Incontrast, the BCL-xL, -xβ and -ΔTM isoforms were equally expressed inboth the viable Hoechst-negative fraction and the apoptoticHoechst-positive fraction of activated T-cells (FIG. 6). Afterstimulation of O3 T cell clone by plate-bound anti-CD3 antibody for 5hours, the O3 cells were subjected to staining with Hoechst 33342 dyeand propidium iodide. The results show that BCL-x γ is selectivelyexpressed in Hoechst-negative cells but not in Hoechst-positive(apoptotic) cells while all other Bcl-x isoforms are expressed in bothforms. Failure to detect BCL-x γ in Hoechst-positive cells did notresult from degraded preparations of total RNA or cDNA, since β-actinand other BCL-x isoforms were detected in these samples. The PCRamplified fragments analyzed on agarose gel were scanned and quantitatedusing an IS-1000 digital imaging system (Alpha Innotech Corp.) followedby normalization, as described above. Failure of BCL-xγ expression afterCD3 ligation represents a genetic marker of apoptosis, while activatedT-cells that express BCL-xγ are spared. The tight coupling of BCL-xγexpression to the TCR may ensure that survival of activated T-cells isgoverned by the nature of TCR engagement rather than by non-specificcytokine stimuli. The observation that BCL-xγ, but not BCL-xL,expression by immature (DP) thymocytes requires host MHC productssuggests that TCR ligation is also necessary for Bcl-xγ expression inthis tissue. Possibly, expression of other isoforms such as BCL-xL maybe important to guarantee survival of immature DP thymocytes long enoughto provide a cellular substrate for positive and negative selection.Expression of BCL-xγ after TCR engagement may be necessary to allowsuccessful positive selection, while failure to induce this gene productmay result in cellular apoptosis and negative selection.

Example 10 BCL-xγ is Involved in Thymocyte Development

Thymocytes from Balb/c and DBA/2 mice were triple stained withflourescent antibodies against CD4, CD8, and Vb6. by flow cytometriccell sorting, CD4⁺CD8^(low) cells for the Vβ6+ subset were collected andanalyzed for expression of β-actin and Bcl-x γ by RT-PCR (FIG. 7).

Thymocytes from C57B1/6 mice were labelled with biotinylated anti-CD69antibody followed by precipitation with streptavidin-conjugatedDynabeads (6×10⁸ beads/ml, beads:target cells 10:1). Separation wasconfirmed by flow cytometry. Fractions were anayzed by RT-PCR forexpression of β-actin and Bcl-x γ. BCL-xγ expression was confined to theCD69⁻ fraction of thymocytes (FIG. 8).

Thymocytes from C57B1/6 mice were fractionated with anti-CD4 andanti-CD8 conjugated Dynabeads (beads:target cells 4:1). The supernatantfraction was separated into two subfractions by biotin anti-CD44 plusstreptavidin-Dynabeads. Separation was confirmed by flow cytometry.Fractions were analyzed by RT-PCR for expression of β-actin and BCL-x γ(FIG. 9). Expression of BCL-x γ was not detected in double negative (DN)(CD4⁻ 8⁻) thymocytes (either CD44⁺ or CD44⁻) from normal, Rag-2^(−/−) orTCRβ^(−/−) donors, nor in single positive (SP) thymocytes (>95%).

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated protein, comprising an amino acid sequence at least 95%homologous to the amino acid sequence of SEQ ID NO: 2 and wherein saidprotein is anti-apoptotic.
 2. An isolated protein, or a portion thereof,wherein the protein comprises amino acids 185–235 of SEQ ID NO: 2 and isanti-apoptotic.
 3. An isolated protein, or portion thereof, comprisingthe amino acid sequence of SEQ ID NO: 2, wherein the protein or portionthereof is anti-apoptotic.
 4. An isolated BCL-xγ fusion proteincomprising the amino acid sequence of SEQ ID NO: 2 or a portion thereof,wherein the amino acid sequence of SEQ ID NO:2 or portion thereof isanti-apoptotic.
 5. An isolated protein, or portion thereof, encoded bythe nucleic acid sequence of SEQ ID NO: 1, wherein the protein orportion thereof is anti-apoptotic.